Ganoderma lucidum polysaccharides (GLP) has been demonstrated to elicit antitumorigenic and proapoptotic activities in cancer; however, the molecular mechanisms underlying the anticancer effects of GLP have yet to be elucidated. Non-steroidal anti-inflammatory drug-activated gene-1 (NAG-1) has been reported to exert proapoptotic effects and therefore, may serve an important role in cancer prevention. The present study aimed to elucidate the molecular mechanism by which GLP stimulates anticancer activity in human prostate cancer (PCa) PC-3 cells. In addition, the role of NAG-1 in GLP-induced cancer inhibition was examined. The results of the present study demonstrated that GLP significantly inhibited cell viability in a time- and dose-dependent manner in PC-3 cells. Flow cytometry indicated that GLP induced late apoptosis, which was accompanied by poly (ADP-ribose) polymerase 1 (PARP) cleavage, and inhibition of pro-caspase-3, -6 and -9 protein expression. Furthermore, it was observed that the expression levels of NAG-1, and its transcriptional factor early growth response-1, were upregulated in a time- and dose-dependent manner upon GLP treatment. The results of a luciferase assay demonstrated that GLP induced the promoter activity of NAG-1, thus indicating that NAG-1 may be transcriptionally regulated by GLP. The secretion of NAG-1 proteins into the cell culture medium was also upregulated upon GLP treatment. Furthermore, inhibition of NAG-1 expression by small interfering RNA significantly, but not completely, prevented GLP-induced apoptosis, and reversed the effects of GLP on PARP and pro-caspase expression. It was further demonstrated that GLP inhibited the phosphorylation of protein kinase B and mitogen-activated protein kinase/extracellular signal-regulated kinase signaling in PC-3 cells. The present study is the first, to the best of our knowledge, to report that GLP may induce apoptosis of PCa cells, which is partially mediated through NAG-1 induction. The present findings may be helpful in elucidating the anticancer mechanisms of GLP through NAG-1 induction for its chemopreventive potential in PCa.