It is unclear how docosahexaenoic acid (DHA) improves insulin resistance via modulating gut microbiome in obese individuals. We used diet-induced obesity (DIO) mice as a model to study the effects of DHA-rich fish oil (DHA-FO) on host metabolic disorders and colonic microbiome. DHA-FO reduced fat deposition, regulated lipid profiles and alleviated insulin resistance in DIO mice. Probably because DHA-FO prevented the permeation of lipopolysaccharide across intestinal epithelial barrier, and promoted peptide YY (PYY) secretion via the mediation of short chain fatty acids receptor (FFAR2) in colon. Furthermore, DHA-FO might regulate PYY expression by reversing microbial dysbiosis, including increasing the abundance of Akkermansia muciniphila and Lactobacillus, and suppressing the growth of Helicobacter. DHA-FO also altered gut microbial function (e.g. "linoleic acid metabolism") associated with PYY expression (r > 0.80, P < 0.05). Herein, DHA-FO enhanced insulin action on glucose metabolism by altering gut microbiome and facilitating colonic PYY expression in DIO mice.