BACKGROUND: The endocannabinoid system regulates various aspects of hepatic fibrosis, however, nothing is known about its role in regulating cholangiocyte proliferation and function. AIM: We evaluated the effects of AEA on cholangiocyte proliferation; and explored the effects of AEA on the thioredoxin1 (TRX1)/redox factor 1 (Ref1)/AP-1 pathway. METHODS: Mice underwent bile duct ligation (BDL) and were infused with anandamide for 3 days post surgery. Proliferation and apoptosis were evaluated in liver sections. Effects of in vitro AEA treatment on cholangiocyte proliferation and apoptosis was studied in purified cholangiocytes. The relative expression of cannabinoid receptors was also assessed in liver sections and cholangiocytes. RESULTS: Cannabinoid receptors, Cb1 and VR1, mRNA expression was decreased after BDL, whereas there was an upregulation of Cb2 mRNA. AEA decreased cholangiocyte growth, and induced accumulation of reactive oxygen species, upregulation of TRX1, Ref1, c-Fos and c-Jun expression, increased nuclear localization of TRX1 and increased AP-1 transcriptional activity. Specific knockdown of TRX1 or Ref1 expression ablated the AP-1 transcriptional activity and AEA-induced cell death, but not expression of c-Fos and c-Jun. Knockdown of c-Fos and c-Jun expression also ablated AEA-induced apoptosis. CONCLUSIONS: AEA suppresses cholangiocyte proliferation during cholestasis via a Cb2-dependent mechanism. Modulation of the endocannabinoid system may be important in the treatment of cholangiopathies.