Egg oil from Portunus trituberculatus (Pt-egg oil) can overcome insulin resistance resulting from abundant bioactive lipids. However, its effects on obesity and gut microbiota were unclear. Here, we evaluated whether Pt-egg oil could improve obesity and gut microbiota or not in high-fat diet feeding mice. Results exhibited that Pt-egg oil markedly reduced body weight and adipose weight gain, improved lipid accumulation and circulatory cytokines, inhibited epididymal adipose cell size. Moreover, Pt-egg oil modified gut microbiota, involving decreases in the ratio of Firmicutes to Bacteroidetes, Proteobacteria, Actinobacteria, and increase in Verrucomicrobia phylum. Pt-egg oil reduced serum and fecal lipopolysaccharide (LPS) levels and down-regulated Toll-like receptor 4 pathway in both epididymal adipose and liver tissues. Meanwhile, Pt-egg oil increased short chain fatty acids and up-regulated of G-protein-coupled receptors in both epididymal adipose and liver tissues. These suggest that Pt-egg oil could be alternative food supplement for the prophylactic effects on anti-obesity and improvement in human gut health.