To investigate the biological meaning of apparent diffusion coefficient (ADC) in tumors following radiotherapy. Five mice bearing TRAMP-C1 tumor were half-irradiated. Diffusion-weighted images were acquired using multiple b-values of up 0 to 3000 s/mm2. The pixelwise ADC positively correlated with extracellular space and nuclear size, and negatively correlated with nuclear count, cytoplasmic space and nuclear space. Optimal ADC was achieved at b-value of 800 s/mm2 in determining the treatment response. Pixelwise ADC values correlate with histology metrics might be a means of in vivo radiomics biomarkers for evaluating tumor heterogeneity and responses to radiotherapy.