Cytochrome P450 (CYP) 3A4, which is abundant in human liver and small intestine and participates in the metabolism of various drugs and xenochemicals, is known to be induced by 1,25-dihydroxyvitamin D 3 (1,25(OH) 2 D 3 ) in the colon carcinoma cell line Caco-2 cells. Nitric oxide (NO) is able to inhibit CYP3A4 expression and catalytic activity. In this study, we investigated the mechanism of suppression by NO of 1,25(OH) 2 D 3 -induced CYP3A4 expression in Caco-2 cells. Caco-2 cells were exposed for 36 h to 400 nM 1,25(OH) 2 D 3 , and the induction of CYP3A4 mRNA expression was detected by real-time PCR. Because c-Myc regulates the expression of several genes, we examined its effect on the CYP3A4 expression induced by 1,25(OH) 2 D 3 . The expression of c-myc mRNA was increased in the early stage but decreased 36 h after the treatment of Caco-2 cells with 1,25(OH) 2 D 3 . The NO donor NOR-4 suppressed CYP3A4 expression induced by 1,25(OH) 2 D 3 in Caco-2 cells in contrast, it significantly induced c-myc gene expression. Treatment of Caco-2 cells with the c-myc antisense oligonucleotide reversed the inhibitory effect of NOR-4 on CYP3A4 expression induced by 1,25(OH) 2 D 3 . These results suggest that the suppression of 1,25(OH) 2 D 3 -induced CYP3A4 expression by NO is due to c-myc expression.