BACKGROUND AND PURPOSE: Tumor suppressor p53 is traditionally recognized as a surveillance molecule to preserve genome integrity.Recent studies have emerged on discovering its functions in metabolic diseases.Here we investigated the role of p53 in the regulation bile acid disposition and cholestasis.EXPERIMENTAL APPROACH: Bile acid disposition related gene expression profile altered by p53 activation was assessed in mouse primary hepatocytes with p53 depletion and in Trp53-null mice.Dual luciferase reporter assay was used to detect the transcriptional activities of target genes.Anticholestatic effects of p53 activator doxorubicin (Dox) were investigated in a 0.5% cholic acid (CA)-fed mouse model of cholestasis.Metabolomic changes of individual bile acids were analyzed using Liquid chromatography/mass spectrometry KEY RESULTS: Dox-mediated p53 activation induced Cyp2b10, Sult2a1 and Abcc2/3/4 expression of mice in vitro and in vivo.Transcriptional activities of CYP2B6 (human ortholog of Cyp2b10) and ABCC3 were directly activated by p53 overexpression.Dox attenuated CA-induced cholestasis in mice, as demonstrated by shrunken gallbladder size, decreased serum total bile acid and total bilirubin levels and ALP activity.Targeted metabolomics analysis revealed that Dox enhanced the excretion of bile acid metabolites from serum and liver to intestine and feces.Upregulation of Cyp2b10, Sult2a1 and Abcc2/3/4 expression was further confirmed in cholestastic mice.p53 deficiency aggregated CA-induced cholestatic injury in mice and bile acid abundance was decreased in intestine and feces.CONCLUSION AND IMPLICATIONS: Our findings suggest a novel role of p53 in promoting bile acid disposition and alleviating cholestastic syndrome, which provides a potential therapeutic target for cholestasis.