Toona sinensis leaf (TSL) has been shown to lower plasma triacylglycerol levels and diminish the size of visceral fat cells in vivo. The molecular mechanism of TSL ethanol extract (TSL-E) on lipid metabolism in 3T3-L1 adipocytes was investigated in this study. Oil Red O staining as well as immunoblotting, real-time PCR, and dual-Luciferase reporter system were performed to investigate the effect of TSL-E on lipid accumulation and the regulation of lipid metabolism, respectively. In addition, active compounds in the TSL-E were analyzed by HPLC. TSL-E significantly decreased lipid accumulation, stimulated free fatty acid (FFA) release, and up-regulated peroxisome proliferator-activated receptor-α (PPARα) and genes involved in peroxisomal (acyl-CoA oxidase) and mitochondrial (uncouple protein 3) fatty acid oxidation. TSL-E also up-regulated cytoplasmic triacylglycerol hydrolysis gene (adipose triglyceride lipase) and genes related to fatty acid oxidation (AMP-activated protein kinase, acetyl-CoA carboxylase, carnitine palmitoyltransferase I, PPARγ, and adiponectin). The major constituents directly inducing PPARα transactivity in TSL-E are gallic acid, rutin, palmitic acid, linoleic acid, and α-linolenic acid. These results indicate that the inhibitory effect of TSL-E on lipid accumulation was through PPARα activation and further up-regulation of PPARα-mediated genes plus up-regulation of cytoplasmic genes involved in lipid catabolism.