It remains to study whether docosahexaenoic acid-rich fish oil (DHA-FO) improves hepatic lipid metabolism by leptin-independent mechanisms. We used ob/ob mice as a model to investigate the effects of DHA-FO on hepatic steatosis. DHA-FO inhibited lipid droplets (LD) formation in liver of ob/ob mice. Probably because DHA-FO consumption prevented the accumulation of oleic acid, and suppressed the synthesis of triglycerides and cholesteryl esters. These beneficial effects might be concerned with the promotion of short chain fatty acids (SCFAs) production. Furthermore, DHA-FO could reverse gut bacteria dysbiosis, including increasing the abundance of SCFAs producers (e.g. Akkermansia and unclassified_Muribaculaceae), and suppressing the proliferation of conditional pathogenic bacteria, such as unclassified_Lachnospiraceae. DHA-FO also promoted colonic microbial function ("Glycerolipid metabolism") associated with lipid metabolism. As a potential ingredient for functional food, DHA-FO reduced LD accumulation, which might be associated with modulation of obesity-linked gut microbiome in ob/ob mice.