The influence of dietary cholesterol (CHOL) and short‐chain fatty acids (SCFA; sodium salts of acetic, propionic and butyric acid, 5:5:2 w/w/w) on growth, organ indices, macronutrient digestibility, and fatty acid composition of Atlantic salmon Salmo salar was investigated. Salmon (initial average weight 0.7 kg) held in seawater (7°C) for 175 days were fed one of six diets: 1, without CHOL/SCFA supplement; 2, with 0.5% SCFA; 3, with 2.0% SCFA; 4, with 1.0% CHOL; 5, with 1.0% CHOL and 0.5% SCFA; 6, with 1.0% CHOL and 2.0% SCFA. Neither SCFA nor CHOL supplements had any significant effects on specific growth rate (SGR), mortality, apparent digestibility coefficients (ADC) of macronutrients, total lipid content. Hepatosomatic index (HSI) was slightly increased in salmon fed the CHOL supplement (P < 0.05). Hepatic CHOL concentration, but not the hepatic CHOL pool, was significantly increased (P < 0.001) by dietary CHOL supplementation. The fatty acid compositions of fillet and gut tissues were not influenced by dietary treatment, while significant effects of CHOL supplements were observed in faeces and liver. Less saturated fatty acids and more mono‐ and poly‐unsaturated fatty acids were excreted with faeces in salmon fed CHOL supplements. Salmon fed CHOL supplements significantly reduced the relative concentration of hepatic palmitic acid (C16 : 0), arachidonic acid (C20 : 4 n‐6) and docosahexaenoic acid (C22 : 6 n‐3), while the contents of oleic acid (C18 : 1 n‐9) and eicosenoic acid (C20 : 1 n‐9) were significantly increased. SCFA did not influence the observed effects of dietary CHOL. The present study shows that dietary CHOL supplements profoundly altered excretion and liver metabolism of individual fatty acids in salmon. The impact of this alteration on physiological performance has not been elucidated.