Background/Aim: Prostate cancer (PCa) diagnosis using patient urine samples represents a non-invasive and more convenient method than the conventional biopsy and prostate-specific antigen (PSA) test. This study intended to identify a biomarker panel to distinguish PCa from benign prostate using urine samples. Materials and Methods: We identified six biomarkers with differential gene expression in 154 PCa and benign prostate specimens. We then determined mRNA expression signature and the diagnostic performance of the 6-biomarker panel in 156 urine samples from patients with PCa and benign disease. Results: The 6-biomarker panel distinguished PCa from benign prostate cases with sensitivity of 80.6%, specificity of 62.9% and area under the curve (AUC) of 0.803 (p<0.0001), whereas serum PSA at 4 ng/ml cutoff had sensitivity of 95.5%, specificity of 20.2% and AUC of 0.521 (p<0.0001). Conclusion: The 6-biomarker panel for use in urine samples was able to distinguish PCa from benign prostate with higher specificity and accuracy than PSA and may be useful in clinical settings.