Septins are a family of GTP-binding cytoskeleton proteins expressed in many solid tumors. Septin 9 (SEPT9) in particular was found overexpressed in diverse carcinomas. Herein, we studied the expression of SEPT9 isoform 1 protein (SEPT9-i1) in human prostate cancer specimens. We utilized immunohistochemical staining to study the expression of SEPT9-i1 protein. Staining level was analyzed in association with clinical characteristics and the pathological Gleason grade and score. Fifty human prostate cancer specimens (42 primary tumors and 8 metastatic lesions) were stained by SEPT9-i1 antibody and analyzed. SEPT9-i1 protein was expressed in prostate cancer cells but absent in normal epithelial cells. The intensity of staining was correlated proportionally to pretreatment prostate-specific antigen (PSA) blood levels and Gleason score (P < 0.05). SEPT9-i1 was highly expressed in all metastatic lesions. A significant assocation between SEPT9-i1 expression and high Gleason score on multivariate linear regression analysis was found. We conclude that SEPT9-i1 is expressed in high-grade prostate tumors suggesting it has a significant role in prostate tumorigenesis and that it could serve as a molecular marker for prostate tumor progression.