Background Recently developed time-dependent diffusion MRI has potential in characterizing cellular tissue microstructures; however, its value in imaging prostate cancer (PCa) remains unknown. Purpose To investigate the feasibility of time-dependent diffusion MRI-based microstructural mapping for noninvasively characterizing cellular properties of PCa and for discriminating between clinically significant PCa and clinically insignificant disease. Materials and Methods Men with a clinical suspicion of PCa were enrolled prospectively between October 2019 and August 2020. Time-dependent diffusion MRI data were acquired with pulsed and oscillating gradient diffusion MRI sequences at an equivalent diffusion time of 7.5-30 msec on a 3.0-T scanner. Time-dependent diffusion MRI-based microstructural parameters, including cell diameter, intracellular volume fraction, cellularity, and diffusivities, were estimated with a two-compartment model. These were compared for different International Society of Urological Pathology grade groups (GGs), and their performance in discriminating clinically significant PCa (GG >1) from clinically insignificant disease (benign and GG 1) was determined with a linear discriminant analysis. The fitted microstructural parameters were validated by means of correlation with histopathologic measurements. Results In the 48 enrolled men, the time-dependent diffusion MRI measurements showed that higher GG was correlated with higher intracellular volume fraction and higher cellularity (intracellular volume fraction = 0.22, 0.36, 0.34, 0.37, and 0.40 in GGs 1-5, respectively; P < .001 at one-way analysis of variance), while lower cell diameter was found at higher GGs (diameter = 23.4, 18.3, 19.2, 17.9, and 18.5 μm in GGs 1-5, respectively; P = .002). Among all measurements derived from time-dependent diffusion MRI, cellularity achieved the highest diagnostic performance, with an accuracy of 92% (44 of 48 participants) and area under the receiver operating characteristic curve of 0.96 (95% CI: 0.87, 0.99) in discriminating clinically significant PCa from clinically insignificant disease. Microstructural mapping was supported by positive correlations between time-dependent diffusion MRI-based and pathologic examination-based intracellular volume fraction (r = 0.83; P < .001). Conclusion Time-dependent diffusion MRI-based microstructural mapping correlates with pathologic findings and demonstrates promise for characterizing prostate cancer. © RSNA, 2022 Online supplemental material is available for this article. See also the editorial by Chatterjee and Oto in this issue.