Lung cancer is one of the most common types of cancer and causes 1.38 million deaths annually, as of 2008 worldwide. Identifying natural anti-lung cancer agents has become very important. Gambogenic acid (GNA) is one of the active compounds of Gamboge, a traditional medicine that was used as a drastic purgative, emetic, or vermifuge for treating tapeworm. Recently, increasing evidence has indicated that GNA exerts promising anti-tumor effects; however, the underlying mechanism remains unclear. In the present paper, we found that GNA could induce the formation of vacuoles, which was linked with autophagy in A549 and HeLa cells. Further studies revealed that GNA triggers the initiation of autophagy based on the results of MDC staining, AO staining, accumulation of LC3 II, activation of Beclin 1 and phosphorylation of P70S6K. However, degradation of p62 was disrupted and free GFP could not be released in GNA treated cells, which indicated a block in the autophagy flux. Further studies demonstrated that GNA blocks the fusion between autophagosomes and lysosomes by inhibiting acidification in lysosomes. This dysfunctional autophagy plays a pro-death role in GNA-treated cells by activating p53, Bax and cleaved caspase-3 while decreasing Bcl-2. Beclin 1 knockdown greatly decreased GNA-induced cell death and the effects on p53, Bax, cleaved caspase-3 and Bcl-2. Similar results were obtained using a xenograft model. Our findings show, for the first time, that GNA can cause aberrant autophagy to induce cell death and may suggest the potential application of GNA as a tool or viable drug in anticancer therapies.