The nuclear receptor-type transcription factor retinoic acid receptor-related orphan receptor α (RORα) is a multifunctional molecule involved in tissue development and cellular function, such as inflammation, metabolism, and differentiation; however, the role of RORα during adipocyte differentiation has not yet been fully understood. Here we show that RORα inhibits the transcriptional activity of CCAAT/enhancer-binding protein β (C/EBPβ) without affecting its expression, thereby blocking the induction of both PPARγ and C/EBPα, resulting in the suppression of C/EBPβ-dependent adipogenesis. RORα interacted with C/EBPβ so as to repress both the C/EBPβ-p300 association and the C/EBPβ-dependent recruitment of p300 to chromatin. In addition to the inhibitory effect on C/EBPβ function, RORα also prevents the expression of the lipid droplet coating protein gene perilipin by peroxisome proliferators-activated receptor γ (PPARγ), acting through the specific mechanism of its promoter. We identified a suppressive ROR-responsive element overlapping the PPAR-responsive element in the perilipin promoter and verified that RORα competitively antagonizes the binding of PPARγ. RORα inhibits PPARγ-dependent adipogenesis along with the repression of perilipin induction. These findings suggest that RORα is a novel negative regulator of adipocyte differentiation that acts through dual mechanisms.