Analysis of the surface architecture of human spermatozoa is a necessary step in the development of new approaches to contraception and resolving the causes of human infertility. In this study we have utilized phage display technology to identify peptides that bind with high affinity to the surface of human spermatozoa. Fifteen- and twelve-mer random peptide phage display libraries were screened against paraformaldehyde-fixed spermatozoa and a number of sperm-binding peptides were identified. One peptide, M6, displayed a high level of affinity for the sperm surface and showed sequence homology with a dominant human ZP3 epitope (hZP 25–33). This peptide bound preferentially to the equatorial and post acrosomal domains of the sperm head and exhibited contraceptive activity by virtue of its capacity to impair the fusion of acrosome-reacted spermatozoa with the vitelline membrane of the oocyte. A similar form of contraceptive activity was also observed within an unrelated peptide, K6, derived from screening the 12-mer library. These results indicate that phage display technology is a powerful tool for developing reagents capable of targeting the human sperm surface, providing insights into the composition of this structure and the identity of targets susceptible to contraceptive attack and pathological disruption.