Adipocyte dysfunction is a major cause of obesity, which is associated strongly with many disorders including psychological and medical morbidities, metabolic abnormalities, and cardiovascular diseases as well as a series of cancers. This study investigated the antiadipogenic activity of scutellarin (1) in 3T3-L1 preadipocytes as well as the underlying molecular mechanisms. It was observed that 1 reduced adipocyte differentiation of 3T3-L1 cells potently, as evidenced by a decrease in cellular lipid accumulation. At the molecular level, mRNA expression of the master adipogenic transcription factors, PPARγ and C/EBPα, was decreased markedly. However, mRNA levels of C/EBPβ, the upstream regulator of PPARγ and C/EBPα, were not decreased by 1. Moreover, a dose-dependent upregulation of PPARα was observed for 1. Computational modeling indicated that 1 can bind to PPARα, γ, and δ each in a distinct manner, while it can activate PPARα only by forming a hydrogen bond with Y464, thus stabilizing the AF-2 helix and activating PPARα. Therefore, these results suggest that 1, a major component of Scutellaria baicalensis, attenuates fat cell differentiation by upregulating PPARα as well as downregulating the expression of PPARγ and C/EBPα, thus showing therapeutic potential for obesity-related diseases.