Low-grade inflammation is usually defined as the chronic production and a low-grade state of inflammatory factors, it often does not have symptoms, and has been associated with neurodegenerative disease, obesity, and diabetes. Omega-3 polyunsaturated fatty acids (n-3 PUFAs) are the precursors of many anti-inflammatory metabolites, such as resolvins and neuroprotectins. It is of interest to study the metabolic profile of endogenous n-3 PUFAs in low-grade inflammatory conditions. To evaluate the protective effects of endogenous n-3 PUFAs on low-grade inflammation with the metabolomics approach, we fed fat-1 mice with an n-6 PUFAs rich diet for a long time to induce a low-grade inflammatory condition. Multi-analysis techniques, including structural analysis using quadrupole time-of-flight mass spectrometry with MS mode, were applied in untargeted metabolomics to search for meaningful metabolites with significant variance in fat-1 mice under low-grade inflammation. Following the untargeted metabolomics screening, several meaningful metabolites were selected which were associated with anti-inflammatory effects generated from endogenous n-3 PUFAs for further analysis. The results revealed that the purine metabolism, fatty acid metabolism and oxidative stress response pathways through insulin resistance were involved in anti-inflammatory mechanisms of n-3 PUFA in low-grade inflammatory conditions. For the first time, this study explored the highlighted pathways as contributors to the anti-inflammatory effects of endogenous n-3 PUFAs in low-grade inflammatory conditions.