Biliary lipid secretion plays an important role in gallstone disease and reverse cholesterol transport (RCT). Using Sr‐bI/Abcg5 double knockout mice (dko), the present study investigated the differential contribution of two of the most relevant transporters: adenosine triphosphate (ATP)‐binding cassette subfamily G member 5 and 8 (ABCG5/G8) and scavenger receptor class B type I (SR‐BI) to sterol metabolism and RCT. Plasma cholesterol levels increased in the following order, mainly due to differences in high density lipoprotein (HDL): Abcg5 ko < wild type < Sr‐bI/Abcg5 dko < Sr‐bI ko. Liver cholesterol content was elevated in Sr‐bI ko only (P < 0.05). In Sr‐bI/Abcg5 dko plasma plant sterols were highest, while hepatic plant sterols were lower compared with Abcg5 ko (P < 0.05). Under baseline conditions, biliary cholesterol secretion rates decreased in the following order: wild type > Sr‐bI ko (−16%) > Abcg5 ko (−75%) > Sr‐bI/Abcg5 dko (−94%), all at least P < 0.05, while biliary bile acid secretion did not differ between groups. However, under supraphysiological conditions, upon infusion with increasing amounts of the bile salt tauroursodeoxycholic acid, Abcg5 became fully rate‐limiting for biliary cholesterol secretion. Additional in vivo macrophage‐to‐feces RCT studies demonstrated an almost 50% decrease in overall RCT in Sr‐bI/Abcg5 dko compared with Abcg5 ko mice (P < 0.01). Conclusion: These data demonstrate that (1) SR‐BI contributes to ABCG5/G8‐independent biliary cholesterol secretion under basal conditions; (2) biliary cholesterol mass secretion under maximal bile salt‐stimulated conditions is fully dependent on ABCG5/G8; and (3) Sr‐bI contributes to macrophage‐to‐feces RCT independent of Abcg5/g8. (Hepatology 2013;)