Administration of the antibacterial drug ampicillin (ABPC) significantly increased hepatic bile acid concentrations. In the present study, we investigated the mechanisms for the elevation of bile acid levels in ABPC-treated mice. Hepatic microsomal cholesterol 7α-hydroxylation and CYP7A1 mRNA level were increased 2.0-fold in ABPC-treated mice despite higher bile acid levels in the liver and small intestinal lumen. A significant change in hepatic small heterodimer partner (SHP) mRNA level was not observed in ABPC-treated mice, whereas a marked decrease in ileal fibroblast growth factor 15 (FGF15) mRNA level was observed (3% of vehicle-treated mice). These phenomena were also observed in mice cotreated with bacitracin/streptomycin/neomycin, which are barely absorbed from the intestine. Primary bile acid contents in the small intestinal lumen were increased in ABPC-treated mice, whereas secondary bile acid, deoxycholic acid (DCA), contents were reduced to below detection limits (<0.01 μmol). In ABPC-treated mice, cotreatment with tauroDCA reversed reductions in ileal FGF15 mRNA level. Ileal SHP mRNA level was, however, not decreased in ABPC-treated mice. ABPC administration to farnesoid X receptor (Fxr)-null mice also decreased ileal FGF15 mRNA levels and secondary bile acid content in the small intestinal lumen. These results suggest that ABPC administration elevates hepatic primary bile acid synthesis, at least in part, through suppression of ileal FGF15 expression.