Aims To investigate the neuromechanisms of perceptual learning treatment in patients with anisometropic amblyopia using functional MRI (fMRI) and diffusion tensor imaging (DTI) techniques. Methods 20 patients with monocular anisometropic amblyopia participated in the study. Both fMRI and DTI data were acquired for each patient twice: before and after 30days' perceptual learning treatment for the amblyopic eye. During fMRI scanning, patients viewed the stimuli with either the sound or amblyopic eye. Changes of cortical activation after treatment were evaluated. In the DTI exams, the fractional anisotropy (FA) values, apparent diffusion coefficient (ADC) values, the voxel numbers of optic radiations (ORs), and the number of tracks were compared between the ipsilateral and the contralateral ORs and also between the previous and posterior scans. Results Remarkable increased activation via the amblyopic eyes was found in Brodmann Area (BA) 17-19, bilateral temporal lobes, and right cingulate gyrus after the perceptual learning treatment. No significant changes were found in the FA values, ADC values, voxel numbers, and the number of tracks after the treatment. Conclusions These results indicate that perceptual learning treatment for amblyopia had a positive effect on the visual cortex and temporal lobe visual areas in patients with anisometropic amblyopia.