The neuropathological hallmark of Parkinson’s disease is the loss of dopaminergic neurons in the pars compacta of the substantia nigra (SNc). The degenerative process starts unilaterally and spreads to the dopaminergic system of both hemispheres. However, the complete characterization of the nigra lesion and the subsequent changes in basal ganglia nuclei activity has not yet been achieved in vivo. The aim of this study was to characterize the time course of the nigral lesion in vivo, using longitudinal T2 relaxometry and diffusion tensor imaging, and the changes in basal ganglia nuclei activity, using manganese-enhanced magnetic resonance imaging, in 6-hydroxydopamine (6-OHDA)-lesioned rats. Our results showed that a unilateral SNc lesion induces bilateral alterations, as indicated by the enhancement of magnetic resonance imaging T2 relaxation times in both the ipsilateral and contralateral SNc. Moreover, axial and radial diffusivities demonstrated bilateral changes at 3 and 14 days after 6-OHDA injection in the pars reticulata of the substantia nigra and cortex, respectively, in comparison to the sham group, suggesting bilateral microstructural alterations in these regions. Unexpectedly, manganese-enhanced magnetic resonance imaging showed decreased axonal transport from the ipsilateral subthalamic nucleus to the ventral pallidum in 6-OHDA-lesioned animals compared with the sham group. These findings demonstrate, for the first time in vivo, the temporal pattern of bilateral alteration induced by the 6-OHDA model of Parkinson’s disease, and indicate decreased axonal transport in the ipsilateral hemisphere.