Anthropogenic stressors activating aryl hydrocarbon (Ah) receptor signaling, including polychlorinated biphenyls, impair the adaptive corticosteroid response to stress, but the mechanisms involved are far from clear. Using Ah receptor agonist (-naphthoflavone; BNF) and antagonist (resveratrol; RVT), we tested the hypothesis that steroidogenic pathway is a target for endocrine disruption by xenobiotics activating Ah receptor signaling. Trout (Oncorhynchus mykiss) were fed BNF (10 mg/kg/day), RVT (20 mg/kg/day) or a combination of both (RBNF) for 5 days, and subjected to a handling disturbance. BNF induced cytochrome P4501A1 (CYP1A1) expression in the interrenal tissue and liver, while this response was abolished by RVT, confirming Ah receptor activation. In control fish, handling disturbance transiently elevated plasma cortisol and glucose levels and transcript levels of interrenal steroidogenic acute regulatory protein (StAR), cytochrome P450 cholesterol side chain cleavage (P450scc) and 11-hydroxylase over a 24 h period. BNF treatment attenuated this stressor-induced plasma and interrenal responses; these BNF-mediated responses were reverted back to the control levels in the presence of RVT. We further examined whether these in vivo impacts of BNF on steroidogenesis can be mimicked in vitro using interrenal tissue preparations. BNF depressed ACTH-mediated cortisol production and this decrease corresponded with lower StAR and P450scc, but not 11-hydroxylase mRNA abundance. RVT eliminated this BNF-mediated depression of interrenal corticosteroidogenesis in vitro. Altogether, xenobiotics activating Ah receptor signaling are steroidogenic disruptors, and the mode of action includes inhibition of StAR and P450scc, the rate-limiting steps in steroidogenesis.