The scavenger receptor class B, type I (SR-BI) is an important player in regulation of mammalian lipid homeostasis. We therefore wanted to study this receptor in Atlantic salmon (Salmo salar L.), which requires a diet with particular high lipid content. We have for the first time cloned and characterized SR-BI from a salmonid fish. The predicted 494 amino acid protein contained two transmembrane domains, several putative N-glycosylation sites, and showed 72% sequence identity with the predicted homolog from zebrafish. SR-BI expression was analyzed by reverse transcription Real-Time PCR in several tissues, and a high relative expression in salmon midgut was detected, which may suggest that SR-BI has a role in uptake of lipids from the diet. We also expressed a construct of salmon myc-tagged SR-BI in salmon TO cells and HeLa cells, which gave a protein of approximately 80 kDa on reducing SDS-PAGE using an antibody against the myc-epitope. Immunofluorescence microscopy analyses of the salmon SR-BI protein in transiently transfected HeLa cells revealed staining in the cell periphery and in some intracellular membranes, but not in the nucleus, which indicated that the salmon protein may be a functional membrane protein. We also observed a high degree of co-localization using an anti-peptide SR-BI antiserum. We found that 20 μg mL⁻¹ insulin up-regulated the SR-BI mRNA levels in primary cultures of salmon hepatocytes relative to untreated cells. Oleic acid, EPA, DHA, or dexamethasone did not affect the relative expression of SR-BI in this liver model system. In conclusion, the salmon SR-BI cDNA encoded a protein with several features common to those of mammalian species. SR-BI gene expression was high in the intestine, which leads us to propose that SR-BI may contribute to the uptake of lipids from the diet.