Abstract Purpose: Prostate cancer remains the second most frequent cause of tumor-related deaths in the Western world. Additional markers for the identification of prostate cancer development and progression are needed. Osteopontin (OPN), which activates matrix metalloproteinases (MMP), is considered a prognostic biomarker in several cancers. “In silico” and experimental approaches were used to determine whether OPN-mediated MMP activation may be a signal of prostate cancer progression. Experimental Design: Pearson correlation coefficients were computed for each OPN/MMP pair across seven publicly available prostate cancer gene expression data sets. Using Gene Set Enrichment Analysis, 101 cancer-related gene sets were analyzed for association with OPN and MMP-9 expression. OPN, MMP-9, MMP-2 tissue inhibitor of metalloproteinase-1 plasma levels, and MMP gelatinase activity were measured by ELISA and zymography in 96 and 92 patients with prostate cancer and benign prostatic hyperplasia, respectively, and 125 age-matched healthy men. Results: Computational analyses identified a significant correlation only between MMP-9 and OPN, and showed significant enrichment scores in “cell proliferation”, “genes constituting the phosphoinositide-3-kinase predictor”, “proliferation signature”, and “tumor metastasis” gene sets in association with both OPN and MMP-9. Plasma analyses revealed a significant increase in OPN and MMP-9 levels and activity in patients with prostate cancer in association with clinical variables (prostate-specific antigen >4 ng/mL and Gleason score >7). Significant correlation between OPN and MMP-9 levels were also observed. Mean plasma levels of OPN and MMP-9 decreased in patients with prostate cancer within 6 months after prostatectomy. Conclusions: The concordant computational and experimental data indicate that the extent of OPN pathway activation correlates with prostate cancer progression.