Effects of the type of dietary non-protein energy source on the size and composition of the total body bile acid pool, on faecal bile acid loss and on bile acid synthesis were investigated in rainbow trout. Two diets were formulated (similar DP:DE ratio) that differed in the inclusion of either maize starch (Starch) or rapeseed oil (Fat) as main non-protein source. Fish were fed to satiation for 44 days. Type of non-protein energy source did not substantially affect the body bile acid pool composition. However, feeding the Starch diet resulted in a larger total body bile acid pool size compared with the Fat diet, and this despite enhanced faecal bile acid loss when feeding the Starch diet that was related to more faeces being produced. Bile acid synthesis in fish fed the Starch diet was more than two times higher compared with fish fed the Fat diet. The difference in body bile acid pool size between diets suggests upregulation of bile acid synthesis in fish fed the Starch diet beyond the level needed to compensate for the higher faecal bile acid loss and/or downregulation of bile acid synthesis in fish fed the Fat diet. The underlying mechanisms for this difference in synthesis need further investigation.