Relative enhanced diffusivity (RED) is a potential biomarker for indirectly measuring perfusion in tissue using diffusion-weighted magnetic resonance imaging (MRI) with 3 b values. To optimize the RED MRI protocol for the prostate, and to investigate its potential for prostate cancer (PCa) diagnosis. Prospective. Ten asymptomatic healthy volunteers and 35 patients with clinical suspicion of PCa. 3T T2 - and diffusion-weighted MRI with b values: b = 0, 50, , 150, , 250, , 400, 800 s/mm2 (values in brackets were only used for patients). Monte Carlo simulations were performed to assess noise sensitivity of RED as a function of intermediate b value. Volunteers were scanned 3 times to assess repeatability of RED. Patient data were used to investigate RED's potential for discriminating between biopsy-confirmed cancer and healthy tissue, and between true and false positive radiological findings. Within-subject coefficient of variation (WCV) to assess repeatability and receiver-operating characteristic curve analysis and logistic regression to assess diagnostic performance of RED. The repeatability was acceptable (WCV = 0.2-0.3) for all intermediate b values tested, apart from b = 50 s/mm2 (WCV = 0.3-0.4). The simulated RED values agreed well with the experimental data, showing that an intermediate b value between 150-250 s/mm2 minimizes noise sensitivity in both peripheral zone (PZ) and transition zone (TZ). RED calculated with the b values 0, 150 and 800 s/mm2 was significantly higher in tumors than in healthy tissue in both PZ (P < 0.001, area under the curve [AUC] = 0.85) and PZ + TZ (P < 0.001, AUC = 0.84). RED was shown to aid apparent diffusion coefficient (ADC) in differentiating between false-positive findings and true-positive PCa in the PZ (AUC; RED = 0.71, ADC = 0.74, RED+ADC = 0.77). RED is a repeatable biomarker that may have value for prostate cancer diagnosis. An intermediate b value in the range of 150-250 s/mm2 minimizes the influence of noise and maximizes repeatability. 2 Technical Efficacy Stage: 1 J. Magn. Reson. Imaging 2019. © 2019 The Authors. Journal of Magnetic Resonance Imaging published by Wiley Periodicals, Inc. on behalf of International Society for Magnetic Resonance in Medicine.