Gamboge is a dry resin secreted from Garcinia hanburryi, and gambogenic acid (GNA) is one of the main active compounds of gamboge. We have previously demonstrated the anticancer activity of GNA in A549 cells and pointed out its potential effects in anticancer therapies. Previous studies reported that GNA induced apoptosis in many cancer cell lines and inhibited A549 tumor growth in xenograft of nude mice in vivo. However, the anticancer mechanism of GNA has still not been well studied. In this paper, we have investigated whether GNA-induced apoptosis is critically mediated by the p38 mitogen-activated protein kinase (MAPK) pathway. Our findings revealed that GNA could induce apoptosis, inhibit proliferation, down-regulate the expression of p38 and MAPK, increase the activations of caspase-9, caspase-3, and cytochrome c release. Furthermore, using SB203580, an adenosine triphosphate-competitive inhibitor of p38 MAPK, inhibit the expression of p-p38 and the experimental results show that it may promote the occurrence of apoptosis induced by GNA. Taken together, these results suggested that up-regulation of the p38 MAPK cascade may account for the activation of GNA-induced apoptosis.