Phytosterols, components of soy-derived lipids, are among the proposed exacerbants of parenteral nutrition–associated cholestasis (PNAC). We investigated whether phytosterols contribute to bile acid (BA)–induced hepatocyte damage by antagonizing a nuclear receptor (NR) critically involved in hepatoprotection from cholestasis, FXR (farnesoid X receptor, NR1H4). In HepG2 cells, stigmasterol acetate (StigAc), a water-soluble Stig derivative, suppressed ligand-activated expression of FXR target genes involved in adaptation to cholestasis (ie BSEP, FGF-19, OSTα/β). Furthermore, StigAc antagonized BA-activated, FXR target genes SHP and BSEP in FXR+/+, but not in FXR−/− mouse hepatocytes. Both Stig and StigAc inhibited BA-activated, FXR-dependent reporter gene expression in transfected HepG2 cells, whereas the most prevalent phytosterol in lipids, β-sitosterol, had no inhibitory effect. Finally, among six ligand-activated NR-ligand binding domains (LBDs) tested, antagonism by StigAc was specific to only two (FXR and PXR, pregnane X receptor, NR1I2). We demonstrate that Stig, a phytosterol prevalent in soy-derived PN lipid solutions, is a potent in vitro antagonist of the NR for bile acids FXR.