The purpose of this work was to demonstrate the feasibility of neurite orientation dispersion and density imaging (NODDI) in characterizing the brain tissue microstructural changes of middle cerebral artery occlusion (MCAO) in rats at 3T MRI, and to validate NODDI metrics with histology. A multi-shell diffusion MRI protocol was performed on 11 MCAO rats and 10 control rats at different post-operation time points of 0.5, 2, 6, 12, 24 and 72 h. NODDI orientation dispersion index (ODI) and intracellular volume fraction (V-ic) metrics were compared between MCAO group and control group. The evolution of NODDI metrics was characterized and validated by histology. Infarction was consistent with significantly increased ODI and V-ic in comparison to control tissues at all time points (P<0.001). Lesion ODI increased gradually from 0.5 to 72 h, while its V-ic showed a more complicated and fluctuated evolution. ODI and V-ic were significantly different between hyperacute and acute stroke periods (P<0.001). The NODDI metrics were found to be consistent with the histological findings. In conclusion, NODDI can reflect microstructural changes of brain tissues in MCAO rats at 3T MRI and the metrics are consistent with histology. This study helps to prepare NODDI for the diagnosis and management of ischemic stroke in translational research and clinical practice.