Multifocal inflammatory leukoencephalopathy (MIL) is a rare syndrome that can occur in patients with colon cancer after chemotherapy with 5-fluorouracil (5-FU) and levamisole. Histologic diagnosis by brain biopsy is most effective, but there can be high surgical risks and technical limitations. Therefore, we introduce a noninvasive diagnostic technique using diffusion tensor imaging (DTI) for patients suspected to have MIL. A 45-year-old woman had been treated for 12 weeks with levamisole and 5-FU for adenocarcinoma of the cecum, when she complained of ataxia, diplopia and left ptosis. Brain MRI showed a nodular enhancing lesion in the left midbrain, using gadolinium enhanced T1-weighted imaging, and scattered multiple white matter lesions throughout the cerebral hemisphere. Instead of a brain biopsy, DTI was performed that revealed reduced fractional anisotropy (FA) and increased radial diffusivity (RD) in the left midbrain lesion compared with the right midbrain denoting demyelination. Levamisole and 5-FU were discontinued, and she received intravenously 1 g of methylprednisolone daily for 5 d. After five weeks, follow-up DTI showed an increased FA and decreased RD, signifying the reversibility. DTI therefore may provide valuable information on diagnosing MIL and assessing the treatment response.