ObjectiveAlthough 27‐hydroxycholesterol (27‐HC) has been reported as a potent regulator of lipid homeostasis, its role in hepatic lipogenesis remains obscure. The present study was designed to investigate the impact of 27‐HC on sterol regulatory element‐binding protein 1 (SREBP‐1) and hepatic steatosis.MethodsIn this study, the 27‐HC level in mice was upregulated by overexpressing CYP27A1 or treating primary hepatocytes with 27‐HC, and then the hepatic lipid accumulation was detected.Results27‐HC inhibited hepatic lipid accumulation and decreased the levels of the mature active form of SREBP‐1. The expression of lipogenic genes, including acetyl coenzyme A carboxylase, fatty acid synthase, stearoyl‐coenzyme A desaturase‐1, and glycerol‐3‐phosphate acyltransferase, were also suppressed after 27‐HC intervention. Furthermore, 27‐HC induced expression of insulin‐induced gene‐2 (Insig‐2), an endoplasmic reticulum protein that prevents SREBP activation, both in vivo and in vitro. The inhibitory effect of 27‐HC on SREBP‐1 activation was absent when Insig‐2 was silenced. Finally, coimmunoprecipitation showed that 27‐HC promoted the binding of Insig‐2 to SREBP‐1.ConclusionsThese studies demonstrated the suppressive effect of 27‐HC on hepatic lipid accumulation and revealed a novel mechanism by which 27‐HC regulates lipogenesis.