This study aimed to investigate the Diffusion-Tensor-Imaging (DTI) potential in the detection of microstructural changes in prostate cancer (PCa) in relation to the diffusion weight (b-value) and the associated diffusion length lD. Thirty-two patients (age range = 50–87 years) with biopsy-proven PCa underwent Diffusion-Weighted-Imaging (DWI) at 3T, using single non-zero b-value or groups of b-values up to b = 2500 s/mm2. The DTI maps (mean-diffusivity, MD; fractional-anisotropy, FA; axial and radial diffusivity, D// and D┴), visual quality, and the association between DTI-metrics and Gleason Score (GS) and DTI-metrics and age were discussed in relation to diffusion compartments probed by water molecules at different b-values. DTI-metrics differentiated benign from PCa tissue (p≤ 0.0005), with the best discriminative power versus GS at b-values ≥ 1500 s/mm2, and for b-values range 0–2000 s/mm2, when the lDis comparable to the size of the epithelial compartment. The strongest linear correlations between MD, D//, D┴, and GS were found at b = 2000 s/mm2and for the range 0–2000 s/mm2. A positive correlation between DTI parameters and age was found in benign tissue. In conclusion, the use of the b-value range 0–2000 s/mm2and b-value = 2000 s/mm2improves the contrast and discriminative power of DTI with respect to PCa. The sensitivity of DTI parameters to age-related microstructural changes is worth consideration.