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Feed additives are routinely used for improving poultry health and production worldwide. Chitosan oligosaccharides (COS) is a relatively new feed additive that is a derivative of chitosan, a non-toxic linear polysaccharide with many biological functions. To investigate the efficacy of COS on gut morphology and serum biochemical profile (lipid, protein, glucose, creatinine and enzymes), a total of 180 day-old broiler chicks (n = 45) were allocated into allocated to 1 of 4 groups: control (T0) and treatment groups (T1, T2 and T3) fed basal diet supplemented with increasing amounts of COS for 35 days. Our findings showed that dietary COS supplementation had positive effects on the body weight and body weight gain in the treatment groups. Villus height and width, ratio of villus height and crypts depth, and tunica mucosa thickness of duodenum increased (P > 0.05) in the treatment groups (T1 and T2), whereas crypts depth of duodenum and cecum were decreased in the treatment groups (T1 and T2) compared to the control group; treatment group 3 (T3), however, was almost similar to the control group. In the case of cecum, tunica mucosa thickness, length and width of mucosal folds showed similar results as duodenum. The population and size of intestinal glands and lymphocytic infiltration in the mucosa were increased considerably in the treatment groups (T1 and T2) than control group. Total serum cholesterol, triglyceride, LDL and VLDL cholesterol levels were significantly lowered in the treatment group 2 (T2) than control group or other COS treated groups (P < 0.05), although HDL cholesterol level was significantly (P < 0.05) higher in all of the treatment groups, but substantially higher in the group treatment groups 1 and 3 (T1 and T3) compared to control group. Total serum protein, albumin, and globulin were gradually increased in all of the treatment groups along with the increasing dose rates. The liver and kidney functions (ALT, AST and creatinine) were not significantly (P > 0.05) affected due to COS supplementation. These results suggest that COS has a dose-dependent response to improve gut morphology and health physiology in broilers; low to medium doses are more favorable for safe poultry production as it enhances growth performance, increases villus surface area, reduces undesirable cholesterols and positively affects the blood glucose and protein levels. These results would be helpful in exploring the efficacy of COS as a potential feed additive for safe poultry meat production, which is a time-demanding issue for the poultry industry from the consumers' point of view.