The antidepressant venlafaxine is detected at parts per billion levels in tertiary-treated municipal wastewater effluent. However, the impact of this serotonin-norepinephrine reuptake inhibitor (SNRI) on non-target aquatic animals is poorly understood. We tested the hypothesis that environmentally relevant levels of venlafaxine disrupt the highly conserved cortisol and glucose response to stress in rainbow trout (Oncorhynchus mykiss). Juvenile trout were exposed to venlafaxine (0, 0.2 and 1.0 μg/L) in a static system with daily renewal for seven days. The fish were then subjected to an acute handling disturbance and sampled either prior to (0 h) or 1, 4 and 24h after stressor exposure. Venlafaxine exposure did not affect the handling disturbance-mediated transient elevation in plasma cortisol levels or target tissue glucocorticoid receptor expression. The drug exposure disrupted the interrenal steroidogenic capacity, including altered handling stressor-mediated changes in mRNA abundances of steroidogenic acute regulatory protein and cytochrome P450 side chain cleavage. The handling stressor-induced transient elevations in plasma glucose levels were significantly reduced in the venlafaxine-exposed fish. This was not accompanied by changes in liver glycogen content, glucose transporter 2 mRNA abundance or the glycolytic capacity, whereas the capacity for gluconeogenesis and amino acid catabolism were enhanced. Venlafaxine also brought about changes in the gill of trout, including enhanced lactate dehydrogenase activity and Na(+)-K(+) ATPase protein expression, while the Na(+)-K(+) ATPase enzyme activity was reduced. Collectively, our results demonstrate that venlafaxine at levels detected in the aquatic environment impacts tissue metabolic capacities and may compromise the adaptive responses to an acute stressor in rainbow trout.