Diffusion tensor imaging (DTI) is a non-invasive magnetic resonance imaging technique that can be used to assess microstructural changes in a brain disease such as Parkinson’s disease (PD). Fractional anisotropy (FA) is a commonly used parameter in DTI analysis, although properties of DTI/FA in analyzing brain modifications in PD have been poorly profiled. This study was thus designed to characterize the detailed time course of FA in the substantia nigra (SN). A rat model of PD was induced by microinfusion of 6-OHDA into the right medial forebrain bundle. DTI was performed before and 3, 7, 14, and 28 days after 6-OHDA lesions. Locomotor behavior and apomorphine-induced rotational behavior were tested the next day after DTI except for the day 3 after lesions. Immunohistochemistry was performed to confirm the loss of tyrosine hydroxylase positive neurons and gliosis. A time-dependent and V-shape change in FA values in the SN was observed in PD rats. Following 6-OHDA lesions, FA values were initially decreased and then recovered. A maximal reduction of FA values was reached at 7 days after lesions. At the same time point, a significant linear correlation between reduced FA values and impaired motor activity was observed. Our results establish the temporal property of DTI and suggest that DTI is a useful tool in evaluating the dopamine lesion in the SN in the early phase of PD. The use of DTI may thus facilitate the early diagnosis of PD in clinical practice.