Curcumin indicates varying sensitivities among individuals. To explore the influence of gut microbiota on the bioavailability and biotransformation of curcumin, a gut microbiota clearance model was established by using ceftriaxone. A total of 20 metabolites were identified and quantified by UPLC-MS/MS. The results indicated that (1) curcumin was predominantly distributed in the gastrointestine, and its distribution in the colon showed a significant positive correlation with the kidney; (2) the disrupted gut microbiota increased the excretion of curcumin in urine and inhibited the biotransformation of bioactive metabolites, such as tetrahydrocurcumin and hexahydrocurcumin; (3) the differences in curcumin metabolism were associated with changes in gut microbiota; (4) an in vitro experiment showed that gut microbiota can enhance the transformation of bioactive metabolites. Therefore, gut microbiota may influence the bioavailability and biotransformation of curcumin. This study enhances our understanding of the metabolism of curcumin by the gut microbiota.