Gut microbiota impacts fish metabolism, nutrient utilization, and health. We know little about how temperature and diet interact with rainbow trout gut microbes. A total of 288 fish (average body weight: 45.6 g) fed four iso‐caloric, ‐lipidic, and ‐nitrogenous diets comprised crude protein (40%) and lipid (20%) manufactured as 100% animal‐based protein (AP) and blend of 50 fish oil (FO)/50 camelina oil (CO), 100% AP and 100% CO, 100% plant‐based protein (PP) and blend of 50 FO/50 CO or 100% PP and 100% CO at 14 or 18°C water temperature. Gut content was analyzed using 16S rRNA gene and shotgun sequencing. Alpha‐diversity did not change significantly. Regardless of diet, Tenericutes, Firmicutes, Proteobacteria, Spirochaetes, Bacteroidetes, and Actinobacteria dominated. At family level, Aeromonadaceae and Enterobacteriaceae dominated at 18°C, while Mycoplasmataceae dominated at 14°C. Moreover, genes relating to amino acid, carbohydrate, fat, and energy metabolisms and fatty acid biosynthesis significantly increased at 18°C. Functional profiles did not vary significantly among diets, except for a higher methionine and cysteine metabolism in fish fed plant ingredient compared animal diet, suggesting trout fed plant‐derived protein mixed with CO could be as effective as those fed fish meals mixed with 50/50 FO and CO.