The inhibitory effects of purified fractions isolated from guava seed polysaccharides (GSPS) including guava seed polysaccharide fraction 1 (GSF1), GSF2, and GSF3 on prostate cancer cells remain unclear. To clarify the anti-prostate cancer potential, GSPS, GSF1, GSF2, and GSF3 were isolated using Sepharose 6B gel filtration chromatography to assay their inhibitory effects on prostate PC-3 cell growth with direct action or indirect immunotherapy using either splenocyte conditioned media (SCM) or macrophage conditioned media (MCM). Correlations between cytokine profiles in the conditioned media and pro-apoptotic gene expression levels in the corresponding treated PC-3 cells were analyzed. Results showed that GSPS, GSF1, GSF2, and GSF3, particularly GSF3, through either direct action or indirect treatments using SCM or MCM, significantly ( < 0.05) inhibited PC-3 cell growth. GSF3 direct treatments increased pro-apoptotic Bax/anti-apoptotic Bcl-2 mRNA expression ratios in corresponding treated PC-3 cells. Either SCM or MCM cultured with GSF3 increased Fas mRNA expression levels in corresponding treated PC-3 cells. Both Th2-polarized and anti-inflammatory cytokine IL-10 either secreted in SCM or MCM were positively correlated with Fas mRNA expression levels in corresponding treated PC-3 cells. Our results suggest that GSF3 is a potent biological response modifier to decrease PC-3 cell growth through inducing apoptosis.