OBJECTIVE: Identification of novel regulators involved in adipose development is important to understand the molecular mechanism underlying obesity and associated metabolic disorders. Through isolation and analysis of a vitamin K epoxide reductase Vkorc1l1 mutant, this study aimed to disclose its function and underlying mechanism in adipose development and to obtain valuable insights regarding the mechanism of obesity. METHODS: A Vkorc1l1 mutation recovered from a forward genetic screen for obesity-related loci in mice was characterized to explore its effects in gene expression, animal metabolism, and adipose development. Adipogenesis was evaluated in both Vkorc1l1 mutant stromal vascular fraction and Vkorc1l1 knockdown preadipocytes. Intracellular vitamin K(2) level and the effect of vitamin K(2) on adipogenesis were tested in primary preadipocytes. RESULTS: Vkorc1l1 mutants displayed a considerably lower fat to body weight ratio, substantially decreased plasma leptin, and significantly underdeveloped white adipose tissue. Adipogenic defects related with Vkorc1l1 deficiency were observed both in vivo and in vitro. Vitamin K(2) could inhibit adipogenesis in stromal vascular fraction. Increased intracellular vitamin K(2) level was detected in Vkorc1l1 mutant preadipocytes. CONCLUSIONS: Vkorc1l1 promotes adipogenesis and possibly obesity. Downregulation of Vkorc1l1 increases intracellular vitamin K(2) level and impedes preadipocyte differentiation.