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Parthenolide (PA) shows promising therapeutic effect on ulcerative colitis and colorectal cancer, and can ameliorate colon inflammation in a gut microbiota-dependent manner. In the present study, an effective UHPLC-QTOF-MS method aided by rat intestinal microbiota biotransformation was applied to characterize 34 metabolites (including a series of characteristic R-Sn-R type organosulfur compounds) of PA for the first time. Noteworthy, organosulfur metabolites PA-S2-PA and PA-S3-PA could react with cysteine to generate H2S via persulfide/polysulfide intermediates and significantly inhibit colonic cancer HT-29 and HCT116 cells viability and pro-inflammatory cytokine IL-6 release. These results indicated that PA and its organosulfur metabolites involved in H2S regulation in intestinal tract, which should be beneficial for a more in-depth understanding of the action mechanism of PA.