The constitutive androstane receptor (CAR) modulates xeno‐ and endobiotic hepatotoxicity by regulating detoxification pathways. Whether activation of CAR may also protect against liver injury by directly blocking apoptosis is unknown. To address this question, CAR wild‐type (CAR^+/+) and CAR knockout (CAR^−/−) mice were treated with the CAR agonist 1,4‐bis[2‐(3,5‐dichloropyridyloxy)] benzene (TCPOBOP) and then with the Fas agonist Jo2 or with concanavalin A (ConA). Following the administration of Jo2, hepatocyte apoptosis, liver injury, and animal fatalities were abated in TCPOBOP‐treated CAR^+/+ but not in CAR^−/− mice. Likewise, acute and chronic ConA‐mediated liver injury and fibrosis were also reduced in wild‐type versus CAR^−/− TCPOBOP‐treated mice. The proapoptotic proteins Bak (Bcl‐2 antagonistic killer) and Bax (Bcl‐2‐associated X protein) were depleted in livers from TCPOBOP‐treated CAR^+/+ mice. In contrast, mRNA expression of the antiapoptotic effector myeloid cell leukemia factor‐1 (Mcl‐1) was increased fourfold. Mcl‐1 promoter activity was increased by transfection with CAR and administration of TCPOBOP in hepatoma cells, consistent with a direct CAR effect on Mcl‐1 transcription. Indeed, site‐directed mutagenesis of a putative CAR consensus binding sequence on the Mcl‐1 promoter decreased Mcl‐1 promoter activity. Mcl‐1 transgenic animals demonstrated little to no acute liver injury after administration of Jo2, signifying Mcl‐1 cytoprotection. In conclusion, these observations support a prominent role for CAR cytoprotection against Fas‐mediated hepatocyte injury via a mechanism involving upregulation of Mcl‐1 and, likely, downregulation of Bax and Bak. (HEPATOLOGY 2006;44:252–262.)