There are resourceful phospholipids in the eggs of the crab, Portunus trituberculatus (Pt-PL). However, their components and bioactivities regarding obesity were unclear. Here, we investigated the composition of Pt-PL and their fatty acids. Moreover, its effects on obesity and gut microbiota were also evaluated in high fat diet (HFD)-fed mice. The results showed that Pt-PL contained 12 kinds of phospholipids, mainly including phosphatidylcholine (PC, 32.28%), phosphatidylserine (PS, 26.51%), phosphatidic acid (PA, 19.61%), phosphatidylethanolamine (PE, 8.81%), and phosphatidylinositol (PI, 7.96%). Polyunsaturated fatty acids (PUFAs) predominated in the fatty acids components of Pt-PL, especially eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). Animal experiments demonstrated that Pt-PL significantly alleviated body weight gain, adipose gain, hepatic gain, fasting blood glucose, serum insulin, lipid levels in serum and the liver, and systematic inflammation in HFD-fed mice. Furthermore, Pt-PL regulated gut microbiota, especially in a dramatic reduction in the ratio of Firmicutes to Bacteroidetes at phylum level, as well as significant amelioration in their subordinate categories. Pt-PL reduced fecal lipopolysaccharide and total bile acids, and elevated fecal short chain fatty acid (SCFA) concentrations, particularly acetate and butyrate. These findings suggest that Pt-PL possesses anti-obesity effects and can alter gut microbiota owing to the abundance of PUFAs. Therefore, Pt-PL may be developed as an effective food supplement for anti-obesity and regulation of human gut health.