Nitric oxide (NO) plays an important role in the pathophysiology of sepsis and septic shock but the mechanism is not well understood. The aim of this study was to investigate the role of NO in the cytochrome P450 (CYP) isozyme activity and the expression of its gene during polymicrobial sepsis. The rats were subjected to polymicrobial sepsis by cecal ligation and puncture (CLP). Aminoguanidine (AG, 100 mg/kg body weight) or N(omega)-nitro-L-arginine methyl ester (L-NAME, 100 mg/kg body weight) was injected intraperitoneally at 0, 3, 6, 10, and 20 h after CLP. The plasma nitrite/nitrate concentration increased 24 h after CLP, and this increase was almost completely abolished by AG and L-NAME. Sepsis increased the serum aminotransferase and lipid peroxidation levels, which were attenuated by AG but augmented by L-NAME. The hepatic concentration of the reduced gluthathione decreased in the CLP rats, which was inhibited by AG but augmented by L-NAME. The total CYP content decreased after CLP, which was restored by AG and L-NAME. The CYP1A1, 1A2, and 2E1 activities, along with their protein levels, decreased 24 h after CLP but these decreases were reversed by AG and L-NAME. The CYP1A1, 1A2, 2B1, and 2E1 mRNA expression levels decreased 24 h after CLP, and L-NAME inhibited this decrease. NO plays a key role in the sepsis-mediated decrease in CYP via the interplay of two different mechanisms: NO-dependent suppression of protein via the enhanced inducible NO synthase, and NO-dependent transcriptional suppression via endothelial NO synthase.