Analysis of brown bullhead (Ameiurus nebulosus) bile by ultra performance liquid chromatography high-resolution mass spectrometry (UPLC/HRMS) revealed a series of bile acids similar to those found in humans. Accordingly, we chose this fish as a model organism to examine the metabolism of obeticholic acid, a bile acid used to treat a number of human liver diseases and the one that has the potential to occur as an environmental contaminant. The taurine and glycine conjugates of obeticholic acid and keto-obeticholic acid were identified, as well as the D-cysteinolic acid conjugate of obeticholic acid, likely a metabolite specific to fish. In addition, metabolites of obeticholic acid (sulphate and glucuronide) and several hydroxy-obeticholic acid derivatives were found, representing typical pathways of primary and secondary steroid metabolism. Brown bullhead exposed to obeticholic acid at a dose of 100 mg/kg gave no overt signs of distress or toxicity.