5-Fluorouracil (5-FU) is one of the widely used chemotherapeutic drugs for various cancer treatments, but its chemo-drug resistance is a major obstacle in clinical settings. The anticancer effects of gambogenic acid (GNA) and its potential mechanisms have been well documented in the past few years. In this study, we determined the synergistic inhibitory effects of GNA and 5-FU on A549 human lung cancer cells. 5-FU combined with GNA inhibited the viability of A549 cells in a concentration-dependent manner. The mitochondrial tolerance of this two-kind of drugs combination treatment was stronger than a single-drug treatment. Combination treatment caused a morphological change of A549 cells. Flow cytometric evidence indicated that the combined treatment caused significant cell death, with the death rate of A549 cells treated with combination drugs showing a time-dependent manner. Furthermore, combination treatment of GNA and 5-FU showed up-regulated of caspase-3, caspase-9, bax, RIP1, apoptosis-inducing factor (AIF), voltage-dependent anion channel (VDAC), cytochrome c and cyclophilin D and down-regulated bcl-2. In conclusion, in addition to the activation of caspase-dependent apoptosis, the combination of GNA and 5-FU might also cause cell death of A549 cells by activating caspase-independent necroptosis. These mechanisms may be due to the toxicity of targeted toxin to mitochondria via the mitochondrial pathway.