Accumulating evidence has suggested that fibroblast growth factor 3 (FGF3) is expressed in breast cancer and correlates with the stage and grade of the disease. In the present study, a specific FGF3-binding peptide (VLWLKNR, termed FP16) was isolated from a phage display heptapeptide library with FGF3. The peptide FP16 contained four identical (WLKN) amino acids and demonstrated high homology to the peptides of the 188-194 (TMRWLKN) site of the high-affinity FGF3 receptor fibroblast growth factor receptor 2. Functional analyses indicated that FP16 mediated significant inhibition of FGF3-induced cell proliferation, arrested the cell cycle at the G0/G1 phase by increasing proliferation-associated protein 2G4, suppressing cyclin D1 and proliferating cell nuclear antigen, and inhibited the FGF3-induced activation of extracellular signal-regulated kinase 1/2 and Akt kinase. Taken together, these results demonstrated that the peptide FP16, acting as an FGF3 antagonist, is a promising therapeutic agent for the treatment of breast cancer.