Cytochrome P450 2A5 (CYP2A5) is a murine orthologue of human CYP2A6 and is predominantly found within the endoplasmic reticulum (ER) of the liver. CYP2A5 differs from its P450 counterparts in that it is induced during liver injury caused by hepatitis, liver cancer, and hepatotoxicity. All of these events induce ER stress and protein malfolding, eventually resulting in apoptosis if not rectified. Previous studies have shown that the oxidized form of dithiothreitol (DTTox) induces CYP2A5 in primary mouse hepatocytes while other ER stressors do not, suggesting a reductive ER environment may be associated with its induction. Recently, bilirubin (BR) has been identified as the first endogenous substrate for CYP2A5. Furthermore, BR and its predecessor heme have also been shown to transcriptionally regulate Cyp2a5 and metabolism of BR by CYP2A5 has been shown to provide partial cytoprotection during BR hepatotoxicity. The purpose of this study was to investigate the mechanism of Cyp2a5 gene regulation by reductive stress and to assess the cytoprotective role of CYP2A5 during reductive ER stress. Our results show that the reducing agent 2-mercaptoethanol induces CYP2A5 in a similar manner to DTTox. We also found that DTTox transcriptionally regulates Cyp2a5 via a mechanism that requires the transcription factor Nrf2. Expression of the BR-conjugating enzyme UGT1A1 is also increased after DTTox treatment. Furthermore, hemin induces mRNA splicing of the ER stress-associated transcription factor XBP-1. These data suggest that heme/BR may be playing a role in ER stress-mediated CYP2A5 expression. Finally, our results indicate that CYP2A5 plays a cytoprotective role during DTTox -induced ER stress by reducing XBP-1 mRNA splicing and blocking caspase-3 cleavage.