High protein distillers dried grains (HPDDG) is a co-product of ethanol production that uses prefractionation technology. A 14-week nutrition trial of HPDDG with rainbow trout (Oncorhynchus mykiss: initial weight = 7.5–7.7 g fish−1) was conducted to evaluate the effect of replacing corn gluten meal (CGM) with four levels of HPDDG (0, 50, 100 and 150 g kg−1). HPDDG is higher in crude protein (481 g kg−1, CP) than typical distillers dried grains with soluble (DDGS) (270 g kg−1). We found that 0–150 g kg−1 HPDDG can be used in diets for rainbow trout. Replacement of CGM by HPDDG did not affect feed utilization efficiency or the health status of fish. The final body weight and phosphorus (P) content of fish increased with increasing HPDDG content in the feed. Although increasing dietary HPDDG levels resulted in a reduction in lysine (Lys) and methionine (Met) digestibility, the digestible CP, including Lys and Met, in the experimental diets remained high (above 90%). Better utilization of CP and P by fish fed dietary HPDDG resulted in less waste excretion by these fish than by those fed the control diet. HPDDG may be considered as an alternative protein source for aquaculture feeds.