Monoglycerides, glycerol monoesters of fatty acids, offer several advantages over organic acids by increasing stability, reducing unpleasant odors, and enabling the gradual release of bioactive substances (i.e., organic fatty acids) throughout the intestine, where they can positively affect intestinal integrity. These properties make monoglycerides attractive nutritional interventions for improving animal health and growth. In this context, three studies were performed to investigate the potential beneficial effects of monoglycerides as a practical strategy to improve disease resistance and overall health in pigs.The first study aimed to evaluate the potential of a monoglyceride blend and zinc glycinate as sustainable methods for improving intestinal mucosal integrity in weaned pigs. In vitro cell culture models (porcine enterocyte cell line [IPEC-J2] and porcine alveolar macrophages [PAM]) were used to assess intestinal barrier and immunomodulatory functions. IPEC-J2 cells treated with 250 and 1,000 μg/mL of monoglycerides had increased (P < 0.05) transepithelial electrical resistance (TEER) values at 48 and 72 h after treatment, while 5 μg/mL of zinc glycinate showed increased (P < 0.0001) TEER values only at 72 h after treatment, compared with the control group. Lipopolysaccharide (LPS) challenge increased (P < 0.05) the production of tumor necrosis factor-alpha (TNF-α) and interleukin-1 beta (IL-1β) from PAM. In the non-challenge group, 50 or 100 μg/mL of monoglycerides stimulated (P < 0.05) TNF-α and IL-1β production from PAM. Treatment with 25 or 100 μg/mL of zinc glycinate also enhanced (P < 0.05) TNF-α production from PAM. In LPS-treated PAM, 1,000 μg/mL of monoglycerides increased (P < 0.05) IL-1β production, while zinc glycinate suppressed (P < 0.0001) the secretion of TNF-α and IL-1β at the doses of 100, 250, and 500 μg/mL. The results from in vitro culture assays indicate that monoglycerides have a beneficial effect on strengthening epithelial barrier function, while zinc glycinate may have strong anti-inflammatory benefits.The second study evaluated the effects of dietary monoglycerides (blend of short- and medium-chain fatty acids; BalanGut™ LS L; BASF SE) on the gut health and immunity of weaned pigs experimentally infected with an enterotoxigenic Escherichia coli (ETEC) F18. Pigs in high-dose zinc oxide (ZNO; 3,000 mg/kg) and antibiotic groups had lower (P < 0.05) severity of diarrhea than control, but the severity of diarrhea was not different between antibiotic and monoglycerides groups. Pigs fed with monoglycerides or ZNO had lower (P < 0.05) serum haptoglobin on d 2 or 5 post-inoculation (PI) than control. Pigs in ZNO had greater (P < 0.05) goblet cell numbers per villus, villus area and height, and villus height-to-crypt depth ratio (VH:CD) in duodenum on d 5 PI than pigs in control, monoglycerides, and antibiotic groups. Pigs supplemented with monoglycerides, ZNO, or antibiotic had reduced (P < 0.05) ileal crypt depth compared with control on d 5 PI, contributing to the increase (P = 0.06) in VH:CD. Consistently, pigs in ZNO expressed the lowest (P < 0.05) TNFa, IL6, IL10, IL12, IL1A, IL1B, and PTGS2 in ileal mucosa on d 5 PI, while no difference was observed in the expression of those genes between ZNO and monoglycerides. Supplementation of ZNO or antibiotic had significant impacts on metabolic pathways in the serum compared with control, particularly on carbohydrate and amino acids metabolism, while limited impacts on serum metabolites were observed in monoglycerides group when compared with control. Pigs supplemented with ZNO had greater (P < 0.05) growth performance than other treatments, but no difference was observed in average daily feed intake between ZNO and monoglycerides groups during the post-challenge period. The second study indicates that supplementation of monoglyceride blend may enhance disease resistance of weaned pigs by alleviating the severity of diarrhea and mitigating intestinal and systemic inflammation, although the effectiveness may not be comparable to high-dose zinc oxide.The third study was conducted to explore the effects of dietary supplementation of organic acids (blend of formic acid, lactic acid, and sodium formate; Acitra G20C, Eastman), monoglycerides (blend of short- and medium-chain fatty acids; Entero-Nova 410C, Eastman), and combination of both acid-based additives on diarrhea, immune responses, and growth performance of ETEC F18-challenged weaned pigs. Supplementation of organic acids, monoglycerides, or their combination significantly reduced (P < 0.05) the frequency of diarrhea compared with control group. ETEC F18 infection increased (P < 0.05) counts of total white blood cells, neutrophils, and lymphocytes on d 5 and 14 PI, compared with d 0. Supplementation of the combination of organic acids and monoglycerides reduced (P < 0.05) the counts of total white blood cells, lymphocytes, and the ratio of neutrophils to lymphocytes, and tended (P < 0.10) to reduce neutrophil count on d 5 PI, compared with control. Pigs fed with monoglycerides had higher (P < 0.05) neutrophil counts than pigs in the control group on d 14 PI. Pigs fed with monoglycerides tended (P < 0.10) to have lower serum granulocyte-macrophage colony-stimulating factor, interleukin-1 alpha, and TNF-α concentrations on d 0, 5, or 14 PI compared with control. However, supplementation of organic acids, monoglycerides, or the combination of both had limited impacts on growth performance throughout the experiment. The third study suggests that dietary supplementation of organic acid blend, monoglyceride blend, and or their combination may alleviate diarrhea, intestinal damage, or inflammation caused by ETEC infection in pigs. Moreover, the potential for synergistic effects related to immune-modulatory effect observed in the combination group requires further investigation. In summary, monoglycerides investigated in the present studies have positive effects on disease resistance and the overall health of weaned pigs under ETEC F18 infection, by improving gut integrity and modulating local and systemic inflammation.