The objective of this study was to evaluate whether α-naphthoflavone (ANF) modulates aryl hydrocarbon receptor (AhR) signaling in rainbow trout ( Oncorhynchus mykiss). AhR and cytochrome P450 1A1 (CYP1A1) protein and mRNA content were used as indictors of AhR signaling. Primary culture of rainbow trout hepatocytes were exposed to different concentrations of ANF (10 − 9 –10 − 5 M), while β-naphthoflavone (BNF 10 − 10 –10 − 6 M) and a combination of ANF and BNF were used to elucidate the impact of ANF on AhR signaling. ANF increased AhR and CYP1A1 protein expression in a concentration-related manner; the maximal induction was about 50% that of BNF. Despite the differences in protein content between ANF and BNF stimulation, the maximal AhR and CYP1A1 mRNA abundance seen with the high concentrations of ANF and BNF were similar. ANF significantly decreased (∼50%) BNF-induced AhR protein expression (only at 10 − 9 M), but not CYP1A1 protein and gene expression. In addition, ANF at a sub-maximal concentration (10 − 7 M) did not affect BNF-induced AhR protein content, but increased the sensitivity of hepatocytes to BNF-mediated CYP1A1 protein expression. Taken together, the mode of action of ANF appears similar to BNF, including modulation of AhR expression and activation of AhR-mediated signaling in rainbow trout hepatocytes. Overall, ANF is not only a partial AhR agonist, but may also modify BNF-mediated AhR signaling in trout hepatocytes.