This study was conducted to evaluate the anti-adipogenic effects of Maclura tricuspidate twig extract (MTE) in 3T3-L1 pre-adipocytes. 3T3-L1 pre-adipocytes were differentiated with adipogenic regents (dexamethsone, 3-isobutyl-methylxanthine, insulin) by incubation for 8 days in the absence or presence of MTE. The accumulation of lipid droplets and triglycerides in cells was dose-dependently suppressed by MTE. MTE caused an increase in the release of adiponectin and inhibited the release of leptin. Consistent with these results, MTE attenuated the protein expression of peroxisome proliferator-activated receptor gamma (PPARγ), CCATT/enhancer binding protein alpha (C/EBPα), fatty acid synthase (FAS) and adipocyte Protein 2 (aP2), all of which are associated with adipogenesis in cells. Treatment with MTE also dose-dependently increased the release of free glycerol. Additionally, MTE increased the protein expression of hormone-sensitive lipase (HSL) and adipose triglyceride lipase (ATGL), which are associated with lipolysis. These results showed that MTE inhibits adipogenesis by regulating the expression of proteins involved in adipogenesis. Therefore, the results suggest that MTE has the potential to be used as a functional ingredient in the food industry to prevent obesity.