Adipocyte differentiation requires the coordinated activities of several nuclear transcription factors. Recently, mitochondria biogenesis was reported to occur during adipocyte differentiation and following treatment with thiazolidinediones in vitro and in vivo. Crif1 is a translational factor for mitochondrial DNA (mtDNA) and is important for transcription of the mitochondrial oxidative phosphorylation (OXPHOS) complex. To investigate the role of OXPHOS in adipogenesis, we analyzed adipocyte differentiation following disruption of Crif1 in vitro and in vivo. The adipose-specific Crif1 knockout mouse had a lower body weight and less fat mass than wild-type mice. Furthermore, adipocytes were smaller and had a dysplastic morphology in the adipose-specific Crif1 knockout mouse. 3T3-L1 adipocytes or adipose-derived stem cells (ADSCs) that lacked Crif1 expressed lower levels of mtDNA-encoded OXPHOS subunits, and adipocyte differentiation was disrupted. Rosiglitazone treatment did not induce adipogenesis or mitochondria biogenesis in Crif1 knockout ADSCs. These results show that mitochondrial OXPHOS and Crif1 are required for rosiglitazone- and hormone-induced adipogenesis.