Cadmium is an endocrine disruptor and inhibits corticosteroid production, but the mechanisms are far from clear. We tested the hypothesis that sublethal exposure to environmentally realistic levels of cadmium impairs cortisol production by disrupting the melanocortin 2 receptor (MC2R) signaling in rainbow trout (Oncorhynchus mykiss). Fish were exposed to sublethal concentrations of cadmium (0.75 or 2.0 μg/L) in a flow-through system for 7 d and subjected to an acute secondary stressor to evoke a cortisol response. Cadmium exposure for 7 d did not affect plasma cortisol concentrations, but head kidney mc2r mRNA levels were higher than in control fish. The cortisol stress performance to a secondary-stressor was attenuated in the cadmium groups, and this corresponded with transient reduction in transcript abundance of mc2r and the gene encoding its accessory protein MRAP1 but not MRAP2 in the head kidney. Furthermore, in vivo cadmium exposure attenuated the adrenocorticotropic hormone (ACTH)-, but not 8-br-cAMP-stimulated cortisol production in head kidney slices ex vivo. This corresponded with reduced transcript abundance of mc2r and mrap1, but not mrap2 in these tissue slices. Also, reporter assays with CHO cells transiently transfected with rainbow trout mc2r and zebrafish mrap1 revealed a dose-independent inhibition in ACTH-stimulated luciferase activity by cadmium. Collectively, waterborne exposure to environmentally realistic concentration of cadmium compromises the stressor-induced cortisol response, and a mode of action involves the disruption of MC2R signaling in rainbow trout.