Asthenozoospermia is a common cause of human male infertility, but the molecular mechanism is not fully understood. With Affymetrix Genechips, ropporin, a component of sperm flagella, was identified by comparing the expression profiles in ejaculated spermatozoa from normozoospermic men and patients with asthenozoospermia. Immunohistochemistry was used to analyze the expression characteristic of ropporin in human testis. Reverse transcription-polymerase chain reaction, Western blotting, and indirect immunofluorescence assay were used to determine the expression of ropporin in ejaculated spermatozoa from normozoospermic and asthenozoospermic men. The results showed that ropporin was predominantly expressed in round spermatids in human testis, and located in the principal piece and the end piece of spermatozoa flagella. The expression level of ropporin was significantly lower in asthenozoospermic men than in normozoospermic controls. These data suggested that ropporin may be involved in sperm motility and its decreased expression may contribute to the low sperm motility in asthenozoospermic patients.