The fungus of Ganoderma is a basidiomycete that has been used as a health-promoting supplement in traditional Asian medicine for centuries, and many pharmacologically active substances of Ganoderma have been characterized. Ganoderma formosanum is a native species of Ganoderma isolated in Taiwan, and our previous studies showed that a polysaccharide fraction, PS-F2, purified from the submerged culture fluid of G. formosanum exhibited immunostimulatory and adjuvant activities in vivo. In the current study, we investigated the antitumor and immunomodulatory effects of PS-F2 in mice bearing CT26 colon adenocarcinoma cells. In both preventive and therapeutic regimens, continuous oral administration of PS-F2 effectively suppressed the growth of CT26 tumor with no adverse effects on the animals’ health. PS-F2 treatment resulted in augmented Th1 and CTL responses, while the accumulation of polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs) was significantly reduced in the spleen. PS-F2 treatment also reduced the serum level of TGF-β and the accumulation of tumor-associated macrophages (TAMs) in the tumor. Taken together, our data show that continuous oral administration of G. formosanum polysaccharides PS-F2 can activate T cell responses while inhibiting the generation of PMN-MDSCs and TAMs, leading to delayed tumor progression.