Start domain-containing protein 3 (Stard3) plays roles in intracellular cholesterol distribution, however, the role of Stard3 in the adipogenesis of 3T3-L1 preadipocytes remains unclear. We demonstrated that Stard3 expression was significantly increased during the adipogenesis of 3T3-L1 preadipocytes, accompanied by an increase of mitochondrial Reactive oxygen species (ROS). Stard3 knocking-down inhibited 3T3-L1 preadipocyte adipogenesis with decreased mitochondrial ROS levels, while ROS inducer rescued the stard3 silencing 3T3 cells with increased ROS. Moreover, Stard3 silencing reduced the expression of peroxisome proliferator-activated receptor-gamma (PPAR gamma) and CCAAT/enhancer binding protein (C/EBP)alpha in 3T3- L1 cells. In conclusion, Stard3 enhanced the adipogenesis of preadipocytes by enhancement of cholesterol redistribution to the mitochondrial, increasing mitochondrial ROS production. These results suggest that Stard3 is an essential factor for the 3T3-L1 cells' differentiation. (C) 2018 Elsevier Inc. All rights reserved.