Aims: Vitamin A and its metabolites has been found to be protective against cholestatic liver injury, but the exact underlying mechanisms involved in cholestatic liver injury remain unclear. The objective of this study was to determine the function and mechanisms of 9-cis-retinoic acid, the metabolite of vitamin A, in cholestatic liver injury. Methods: The bile duct ligated (BDL) mice were treated with 9-cis-retinoic acid by intravenous injection through the tail for 10 days. The liver function and histology were assessed in the matched group and experimental group. The expression of MRP3 in liver tissue was tested by qRT-PCR, Western blotting, and IHC. Effect of RXR alpha sumoylation on MRP3 expression was investigated at a cellular level. Influence of 9-cis-retinoic acid on RXR alpha sumoylation was also tested in cells. Results: Our findings showed that 9-cis-retinoic acid significantly decreases the serum ALT and AST level, alleviates hepatic necrosis of the BDL-mice. We also identified MRP3, an important protective hepatobiliary transporter in cholestasis, was elevated by 9-cis-retinoic acid in vivo and in vitro. 9-cis-retinoic acid weakened the sumoylation of RXR alpha, which promotes the cytoplasmic location of RXR alpha and lightens the interaction of RXR alpha and RAR alpha. Inhibition of RXR alpha and RAR alpha interaction increased MRP3 expression. Conclusions: 9-cis-retinoic acid alleviates cholestatic liver injury by elevating MRP3 expression through its mechanism of inhibiting sumoylation of RXR alpha. (C) 2018 Elsevier Inc. All rights reserved.