Previous studies have shown that fibroblast growth factor 8b (FGF8b) is up-regulated in a large proportion of prostate cancer, and plays a key role in prostate carcinogenesis. In this study, we first designed and synthesized a gN helix domain derived short peptide (named 8b-13) based on the FGF8b-FGFR structure analysis. The synthetic peptides inhibited proliferation of prostate cancer cell lines including PC-3 and DU-145 cells. Further investigations indicated that 8b-13 arrested the cell cycle at the G0/G1 phase, reduced the activations of Erk1/2, P38, and Akt cascades, and down-regulated the expression of G1/S-specific cyclinD1. Suppression of DNA synthesis and G1 to S phase transition caused by regulating the expressions of proteins related to proliferation and cell cycle progression may partly contribute to the inhibitory effect of 8b-13 peptides on the cellular proliferation. Our results not only suggest 8b-13 has a potential antitumor effect on prostate cancer, but also confirm the essential role of the gN helix domain in mediating the activities of FGF8b.