Objective To investigate the effects of n-3 polyunsaturated fatty acids (n-3 PUFAs) on the gut microbiota in diet-induced obese (DIO) mice. Methods Thirty 3~4-week-old male C57BL/6J mice were randomly divided into 3 groups and fed 4 months with two different high-fat diets (34.9% of fat providing 40% of total energy)- the high-fat diet (lard oil and sunflower oil) and the fish oil high-fat diet (lard oil and fish oil),and a low-fat diet (4.3% of fat from lard oil and sunflower oil,providing 10% of total energy).The stool bacteria were examined by 16s DNA real-time PCR,and the mRNA expression levels of IL-1β,IL-6,IL-10,TNF-α and MCP-1 in the colon were determined by reverse transcription real-time PCR. Results As compared to the lean mice,the obese mice induced by the high-fat diet had more Firmicutes and Lactobacillus,and less bacteroidetes,actinobacteria,proteobacteria and bifidobacterium in stool.Comparing differences between the two groups of obese mice,higher abundance of stool bifidobacterium and lower lactobacillus were found in mice with the fish oil high-fat diet.Analysis for mRNA expressions of inflammatory factors in the colon showed that IL-1β,IL-6,TNF-α and MCP-1 were all increased in the high-fat diet induced mice compared to the mice fed low-fat diet,and fish oil n-3 PUFAs reduced expressions of colon IL-1β and TNF-α. Conclusion Fish oil n-3 PUFAs may have beneficial effects on gut microbiota and related colon inflammation in DIO mice.