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Drug‐induced liver injury (DILI) is one of the common adverse effects of drug therapy, which is closely associated with oxidative stress, apoptosis, and inflammation response. Medium‐chain fatty acids (MCFA) were reported to relieve inflammation and attenuate oxidative stress. However, little has been known about the hepatoprotective effects of MCFA in DILI. In the present study, acetaminophen (AP) and rifampicin (RFP) were used to establish DILI models in LO2 cells, and the cytoprotective effects of MCFA on hepatocellular injury were investigated. Results showed that the optimal condition for the DILI model was treatment with 10 mM AP or 600 µM RFP for 24 hr. LCFA treatment markedly reduced the cell viability and increased the activities of alanine aminotransferase, aspartate aminotransferase, and lactate dehydrogenase. Meanwhile, LCFA treatment aggravated cell apoptosis, mitochondrial dysfunction, and oxidative stress. The mRNA and protein expression levels of inflammatory cytokines (IL‐1β and TNF‐α) were significantly elevated by LCFA. In contrast, MCFA treatment did not significantly affect cell viability, apoptosis, oxidative, stress and inflammation, and it did not produce the detrimental effects on DILI models. Therefore, we proposed that MCFA may be more safe and suitable than LCFA as nutrition support or the selection of daily dietary oil and fat for the patients with DILI., In this study, we investigated the optimal conditions for establishing the DILI model and the cytoprotective effects of MCFA on the model. Our results found that MCFA decreased the percentages of apoptotic cells, mitochondrial membrane potential as well as attenuated oxidative stress compared with LCFA. MCFA also down‐regulated the mRNA and protein expression of inflammatory cytokines.