Background and Aims: Bile acids trigger a hepatic inflammatory response, causing cholestatic liver injury. Runt-related transcription factor-1 (RUNX1), primarily known as a master modulator in hematopoiesis, plays a pivotal role in mediating inflammatory responses. However, RUNX1 in hepatocytes is poorly characterized, and its role in cholestasis is unclear. Herein, we aimed to investigate the role of hepatic RUNX1 and its underlying mechanisms in cholestasis. Approach and Results: Hepatic expression of RUNX1 was examined in cholestatic patients and mouse models. Mice with liver-specific ablation of Runx1 were generated. Bile duct ligation and 1% cholic acid diet were used to induce cholestasis in mice. Primary mouse hepatocytes and the human hepatoma PLC/RPF/5-ASBT cell line were used for mechanistic studies. Hepatic RUNX1 mRNA and protein levels were markedly increased in cholestatic patients and mice. Liver-specific deletion of Runx1 aggravated inflammation and liver injury in cholestatic mice induced by bile duct ligation or 1% cholic acid feeding. Mechanistic studies indicated that elevated bile acids stimulated RUNX1 expression by activating the RUNX1-P2 promoter through JAK/STAT3 signaling. Increased RUNX1 is directly bound to the promotor region of inflammatory chemokines, including CCL2 and CXCL2, and transcriptionally repressed their expression in hepatocytes, leading to attenuation of liver inflammatory response. Blocking the JAK signaling or STAT3 phosphorylation completely abolished RUNX1 repression of bile acid–induced CCL2 and CXCL2 in hepatocytes. Conclusions: This study has gained initial evidence establishing the functional role of hepatocyte RUNX1 in alleviating liver inflammation during cholestasis through JAK/STAT3 signaling. Modulating hepatic RUNX1 activity could be a new therapeutic target for cholestasis.