Triterpenes isolated from Ganoderma lucidum could inhibit the growth of numerous cancer cell lines and were thought to be the basis of the anticancer effects of Ganoderma lucidum. Ganoderic acid D (GAD) is one of the major components in Ganoderma triterpenes. GAD treatment for 48 h inhibited the proliferation of HeLa human cervical carcinoma cells with an IC50-value of 17.3 ± 0.3 µM. Flow cytometric analysis and DNA fragmentation analysis indicated that GAD induced G2/M cell cycle arrest and apoptosis. To identify the cellular targets of GAD, 2-DE was performed and proteins altered in expressional level after GAD exposure of cells were identified by MALDI-TOF MS/MS. The regulation of proteins was also confirmed by Western blotting. The cytotoxic effect of GAD was associated with regulated expression of 21 proteins. Furthermore, these possible GAD-target related proteins were evaluated by an in silico drug target searching program INVDOCK. The INVDOCK analysis results suggested that GAD could bind 6 isoforms of 14-3-3 protein family, annexin A5 and aminopeptidase B. The direct binding affinity of GAD towards 14-3-3 zeta was confirmed in vitro using Surface Plasmon Resonance (SPR) Biosensor Analysis. In addition, the intensive study of functional association among these 21 proteins revealed that 14 of them were closely related in the protein-protein interaction network. They had been found to either interact with each other directly, or associate with each other via only one intermediate protein from previous PPI experimental results. When the network was expanded to a further interaction outward, total 21 proteins can fall into one network. In this way, the possible network associate with GAD-target related proteins was constructed and the possible contribution of these proteins to the cytotoxicity of GAD was discussed in this paper.