Commensal bacteria-derived metabolites are critical in regulating the host immune system. Although the impact of gut microbiota-derived hydrophilic metabolites, such as short-chain fatty acids, on immune cell functions and development has been well documented, the immunomodulatory effects of gut microbiota-derived lipids are still of interest. Here, we report that lipid extracts from the feces of specific-pathogen-free (SPF), but not germ-free (GF), mice showed regulatory T (Treg)-cell-inducing activity. We conducted RP-HPLC-based fractionation and liquid chromatography–tandem mass spectrometry (LC–MS/MS)-based lipidome profiling and identified two bioactive lipids, 9,10-dihydroxy-12Z-octadecenoic acid (9,10-DiHOME) and all-transretinoic acid (atRA), with Treg-inducing activity in vitro. The luminal abundance of 9,10-DiHOME in the large intestine was significantly decreased by dextran sulfate sodium (DSS)-induced colitis, indicating that 9,10-DiHOME may be a potential biomarker of colitis. These observations implied that commensal bacteria-derived lipophilic metabolites might contribute to Treg development in the large intestine.