Our previous in vitro study demonstrated that the rat hyperplasia suppressor gene (rHSG) inhibited the proliferation of C6 cells. In the present study, we investigated further the effects of rHSG overexpression on the apoptosis of C6 cells and the possible pathways involved. Hoechst 33342/PI double staining and comet assay were used to examine the morphological characteristics of apoptosis and to examine the effects of rHSG on the apoptosis of the C6 cells. Western blot analysis was used to determine the effects of rHSG overexpression on the protein expression levels of poly(ADP‑ribose) polymerase (PARP), cleaved caspase-3, phosphorylated extracellular signal-regulated kinase 1/2 (p-Erk1/2), phosphorylated Akt (p-Akt) and phosphoinositide 3-kinase (PI3K)/Akt, as well as on the mitogen-activated protein kinase (MAPK) pathways induced by insulin‑like growth factor (IGF)-1. Our results revealed that the C6 cells transfected with the rHSG adenoviral vector (Adv-rHSG-GFP group) efficiently expressed rHSG protein; Hoechst 33342/PI double staining and comet assay revealed that rHSG increased C6 cell apoptosis and induced DNA damage. Western blot analysis indicated that rHSG overexpression significantly increased the level of full-length PARP at 24 and 72 h (P<0.01), but decreased the level at 48 h following transfection (P<0.01), while the proteins levels of cleaved PARP and cleaved caspase-3 increased significantly (P<0.01). The protein expression of p-Erk1/2 and p-Akt began to decrease at 48 h post-transfection (P<0.01). In addition, the protein levels of Akt and Erk1/2 induced by IGF-1 were significantly inhibited. On the whole, the findings of the present study demonstrate that rHSG overexpression induces the apoptosis of rat glioma cells, and that these effects may involve the PI3K/Akt and MAPK pathways.