We used a factorial design to determine the effects of dietary carbohydrate content (0.1 or 25.6%) and temperature (10 or 15 °C) on the chronic toxicity of waterborne copper (0 or 144 μg Cu∙L−1) to rainbow trout (Salmo gairdneri) over a 12-wk period. Relative to those on the low-carbohydrate (LC) diet, trout reared on the high-carbohydrate (HC) diet developed enlarged, glycogen-filled livers and attained reduced final mean wet body weights. This response was more pronounced at 10 than at 15 °C. Whereas Cu exposure had no significant effect on the growth or mortality of either LC or HC fish at 15 °C, or LC fish at 10 °C, HC fish at 10 °C showed significantly reduced final weight and increased mortality. Trends toward reduced liver glycogen and increased serum glucose, characteristic of chronic stress, were apparent in all Cu-exposed fish. In Cu-free water, mean Cu concentrations in liver tissue of HC fish were significantly lower than those in LC fish at both temperatures. Copper exposure resulted in significant increases in liver Cu concentration for all diet treatments except HC at 10 °C. Elevated serum sorbitol dehydrogenase levels, indicative of hepatic damage, were apparent in Cu-HC fish at 15 °C and in HC, Cu-HC, and Cu-LC fish at 10 °C. No treatment effects on either serum protein or hematocrit were evident. The treatments during the growth period affected subsequent lethal tolerance of Cu. While the 96-h Cu LC50 was not significantly altered by diet alone at 15 °C, it was elevated in HC fish, relative to LC fish at 10 °C. The Cu LC50 of Cu preexposed fish was significantly elevated, relative to parallel groups held in Cu-free water, for LC fish at both 10 and 15 °C. This acclimation to Cu was not apparent in HC fish at either temperature: no significant elevation in LC50 occurred as a result of preexposure. We conclude that increased dietary carbohydrate can significantly increase the chronic toxicity of Cu to trout, and that the impact is enhanced by reduced water temperature.