Chondroitin sulfate is a structurally diverse glycosaminoglycan, which contains a variable degree of sulfation that helps to determine its biological function. It is involved in the regulation of cellular activity and has been implicated in carcinogenesis. To determine if the non-sulfated chondroitin backbone has a functional role in prostate cancer, we analyzed its expression by immunohistochemistry using the 1B5 monoclonal antibody and a set of tissue microarrays constructed with 227 prostate specimen cores from 81 cases of benign prostate tissue and 77 cases of prostate cancer, of which 69 of these cases are matched. Non-sulfated chondroitin was found in the secretory epithelial cells and stromal regions of both prostatic adenocarcinoma and benign prostatic tissues, as well as in the basal cells of benign glands. A higher percentage of cancerous cells were stained positively for non-sulfated chondroitin as compared with benign secretory cells of the same patient. Cancerous cells stained more intensely for non-sulfated chondroitin. This increase in percentage of cells stained and increase in staining intensity were associated with higher pathological T stage and extraprostatic extension. Non-sulfated chondroitin expression (either staining intensity or percentage of cells stained) in adenocarcinoma and its peritumoral stroma correlated significantly with several clinicopathological parameters of unfavorable outcome, including higher pathological T stage and Gleason score, presence of tumor in both prostatic lobes, extraprostatic extension, seminal vesicle involvement and preoperative prostate-specific antigen levels. These data suggest that non-sulfated chondroitin is a potentially useful biomarker for prostate cancer, and may be involved in regulating prostate cancer behavior.