Polysaccharides were one of the main components of Ganoderma lucidum, a medicinal mushroom well known for its immuno-modulation effects. In the present study, we demonstrated that polysaccharides extracted from Ganoderma lucidum spores (GL-SP) could stimulate splenic mononuclear cells (MNCs) proliferation and cytokine production. To identify the possible cellular targets of GL-SP in MNCs, two-dimensional gel electrophoresis (2-DE)-based comparative proteomics was performed and proteins altered in expressional level after GL-SP treatment were identified by MALDI-TOF MS/MS. Ten proteins with >2-fold increase or decrease expression in GL-SP-treated MNCs compared with control were found and further identified by MALDI-TOF MS/MS analysis and database searching. In the GL-SP-treated MNCs, there were increases in the expression of myosin regulatory light chain 2-A, Rho GDP dissociation inhibitor beta, T-cell-specific GTPase, phosphatidylinositol transfer protein alpha, and decreases in the expression of apoptosis-associated speck-like protein containing a CARD, 14-3-3 tau, beta-actin, tubulin alpha 2, copine I, and gamma-actin. The results of the present study help to provide insight into the immuno-modulating activities of GL-SP.