Endotoxemia is a causal factor in the development of alcoholic liver injury. The present study aimed at determining the interactions of ethanol with different fat sources at the gut-liver axis. Male Sprague Dawley rats were pair-fed control or ethanol liquid diets for 8 weeks. The liquid diets were based on the Lieber-DeCarli formula, with 30% total calories derived from corn oil (rich in polyunsaturated fatty acids). To test the effects of saturated fats, corn oil in the ethanol diet was replaced by either cocoa butter (CB, rich in long chain saturated fatty acids) or medium chain triglycerides (MCT, exclusively medium chain saturated fatty acids). Ethanol feeding increased hepatic lipid accumulation and inflammatory cell infiltration, and perturbed hepatic and serum metabolite profiles. Ethanol feeding with CB or MCT alleviated ethanol-induced liver injury and attenuated ethanol-induced metabolic perturbation. Both CB and MCT also normalized ethanol-induced hepatic macrophage activation, cytokine expression and neutrophil infiltration. Ethanol feeding elevated serum endotoxin level, which was normalized by MCT but not CB. In accordance, ethanol-induced downregulations of intestinal occludin and ZO-1 were normalized by MCT but not CB. However, CB normalized ethanol-increased hepatic endotoxin level in association with upregulation of the endotoxin detoxifying enzymes argininosuccinate synthase 1 (ASS1). Knockdown ASS1 in H4IIEC3 cells resulted in impaired endotoxin clearance and upregulated cytokine expression. These data demonstrate that the protection of saturated fats against alcohol-induced liver injury occur via different actions at the gut-liver axis and are chain length dependent.