Cytochrome P450 2a5 (Cyp2a5) is distinct from other P450 enzymes in that it is induced in the endoplasmic reticulum (ER) of mouse hepatocytes in conditions that are injurious to the liver. These conditions cause ER stress eventually resulting in apoptosis if not rectified. We previously showed that mouse hepatic Cyp2a5 is induced during reductive ER stress caused by the intramolecular disulfide form of dithiothreitol, trans-4,5-dihydroxy-1,2-dithiane (DTT_ox), and that overexpression of Cyp2a5 provides partial protection against apoptosis due to bilirubin (BR), a compound known to cause ER stress. The purpose of this study was to investigate the mechanism of Cyp2a5 gene regulation by DTTox and to determine if Cyp2a5 plays a cytoprotective role during reductive ER stress. Exposure to DTT_ox (10 mM) and another reductive ER stressor, 2-mercaptoethanol (1 mM), for 48 h markedly increased Cyp2a5 protein levels in primary mouse hepatocytes. In addition, DTT_ox transactivated Cyp2a5 via a mechanism involving the transcription factor nuclear factor-(erythroid-derived 2)-like 2 (Nrf2). Expression of the BR-conjugating enzyme, UDP glucuronosyl transferase 1A1 (UGT1A1) was also increased after DTT_ox treatment, however, this was reduced by Cyp2a5 overexpression. Hemin, a porphyrin inducer of Cyp2a5, induced mRNA splicing of X-box binding protein 1 (XBP-1), a transcription factor involved in the ER stress response, however, this was also reduced by Cyp2a5 overexpression. Finally, overexpression of Cyp2a5 partially blocked DTT_ox-mediated caspase-3 cleavage in Hepa 1–6 cells suggesting a cytoprotective role during ER stress. These findings demonstrate that Nrf2-mediated induction of Cyp2a5 in a reducing ER environment provides partial protection against ER stress-induced apoptosis by decreasing XBP-1 mRNA splicing and caspase-3 cleavage.