Triterpenes are the main components with cytotoxicity in Ganoderma lucidum, which is used popularly as a complementary treatment for cancer therapy in traditional Chinese medicine. To investigate the possible interaction between chemotherapeutic agents and triterpenes extracted from G. lucidum, the cytotoxicity of doxorubicin (DOX) combined with Ganoderma triterpenes (GTS) or lucidenic acid N (LCN), a purified compound, was examined in HeLa cells. The combinations targeting DOX with GTS or LCN resulted in a synergistic interaction in HeLa cells. Moreover, to identify the molecular targets of GTS, two-dimensional gel electrophoresis-based comparative proteomics was carried out and proteins with altered expression levels after GTS treatment in HeLa cells were identified by matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry. The results of our proteomic study indicated that the GTS treatment caused regulated expression of 14 proteins, which play important roles in cell proliferation, the cell cycle, apoptosis, and oxidative stress. Flow cytometric analysis confirmed that GTS could induce weak G(0)-G(1) phase arrest and combined use of GTS with DOX could induce apoptosis in cells. Furthermore, GTS enhanced the reactive oxygen species (ROS)-producing effect of DOX, and a ROS scavenger could affect the synergism between GTS and DOX. In cells with high Ku80 protein expression, the synergism between GTS and DOX was also partly affected. Importantly, in cells with high Ku80 expression that were treated with a ROS scavenger, the synergism between GTS and DOX totally disappeared. These results suggest that the synergism between GTS and DOX might be based on GTS-induced sensitization of cells to chemotherapeutics through enhanced oxidative stress, DNA damage, and apoptosis.