Health benefits of GABA and GABA-enriched foods via anti-obesity activity have been demonstrated in animals. However, the functional roles of GABA especially as non-neurotransmitter in adipocytes have not been thoroughly clarified. Thus, this study aimed to assess the impact of GABA on adipogenesis and lipolysis using 3T3-L1 adipocytes. Oil Red O staining was used to assess lipid accumulation and glycerol release was determined as an indicator of lipolysis. The results showed that treatment with GABA (100-500 µM) during adipocyte maturation decreased lipid accumulation by 20-30%. GABA clearly induced the release of glycerol in a dose-dependent manner within 4 h of treatment, and GABA at 500 µM increased glycerol release about two-fold relative to control. GABA-mediated lipolysis depended on both GABA-A and GABA-B receptors as well as on β-adrenergic receptors. The findings suggest that GABA exhibits non-neuronal functions by promoting lipolysis and by suppressing fat accumulation in adipocytes, which potentially support anti-obesity activity of GABA and GABA-enriched products.