Gambogic acid (GA) is the dry resin of Garcinia hanburyi (Guttiferae) with potent anti-tumor activity, various bioactivities, including detoxification, homeostasis, anti-inflammatory and parasiticide, whereas the effect of this natural compound on cancer cells has not been clearly clarified. Here, we examined cellular cytotoxicity by cell viability assay and DNA fragmentation by DNA ladder assay. Activation of different protein expressions were detected by western blot analyses. We first demonstrated that GA reduces the human SH-SY5Y neuroblastoma cell viability with IC50 of 1.28 μM at 6 h which has less toxicity in fibroblast cells. However, lower concentration GA significantly downregulated the expression of anti-apoptotic protein including Bcl-2, Bcl-xL and Mcl-1 which also dramatically activated cleaved caspase-9 and-3 in a dose- and time-dependent manner. Consequently, GA-induced cytotoxicity was not mediated by the Fas/FasL and PI3K/AKT/GSK-3β signaling pathway. In addition, GA-induced cells showed damage morphology which had become cell rounding, neurite retraction, membrane blebbing and shrunken in a dose- and time-dependent manner that clearly indicates this morphological change might be due to the process of apoptosis which shows fragmented DNA. Therefore, the findings presented in this study demonstrate that apoptotic effects of GA on SH-SY5Y cells are mediated by intrinsic mitochondrian-dependent caspase pathway which suggests this natural compound might be effective as an anti-cancer agent for neuroblastoma malignancies.