We examined the microRNA (miRNA) expression profile of 40 prostatectomy specimens from stage T2a/b, early relapse and non-relapse cancer patients, to better understand the relationship between miRNA dysregulation and prostate oncogenesis. Paired analysis was carried out with microdissected, malignant and non-involved areas of each specimen, using high-throughput liquid-phase hybridization (mirMASA) reactions and 114 miRNA probes. Five miRNAs (miR-23b, -100, -145, -221 and -222) were significantly downregulated in malignant tissues, according to significance analysis of microarrays and paired t-test with Bonferroni correction. Lowered expression of miR-23b, -145, -221 and -222 in malignant tissues was validated by quantitative reverse transcription (qRT)-PCR analyses. Ectopic expression of these miRNAs significantly reduced LNCaP cancer cell growth, suggesting growth modulatory roles for these miRNAs. Patient subset analysis showed that those with post-surgery elevation of prostate-specific antigen (chemical relapse) displayed a distinct expression profile of 16 miRNAs, as compared with patients with non-relapse disease. A trend of increased expression (>40%) of miR-135b and miR-194 was observed by qRT-PCR confirmatory analysis of 11 patients from each clinical subset. These findings indicate that an altered miRNA expression signature accompanied the prostate oncogenic process. Additional, aberrant miRNA expression features may reflect a tendency for early disease relapse. Growth inhibition through the reconstitution of miRNAs is potentially applicable for experimental therapy of prostate cancer, pending molecular validation of targeted genes.