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HIV preferentially affects white matter in the brain. Although combination antiretroviral therapy (cART) reduces HIV viral load within the brain, continued inflammation can persist. We investigated the effect of HIV and cART on white matter integrity.We used diffusion tensor imaging (DTI) to examine the effects of HIV and cART on white matter integrity within the corpus callosum and centrum semiovale (CSO).Neuropsychological testing and DTI measures (fractional anisotropy, mean diffusivity, axial diffusivity, radial diffusivity) were obtained from 21 HIV-uninfected controls, 21 HIV-infected patients naive to cART (HIV+/cART−), and 21 HIV+ patients receiving stable cART (HIV+/cART+). A subset of the HIV+/cART− individuals (n = 10) was assessed before and 6 months after receiving medications. Differences among the cross-sectional groups were assessed using an analysis of variance, whereas paired t-tests evaluated longitudinal changes.HIV+/cART− participants had significantly lower mean diffusivity, axial diffusivity, and radial diffusivity for the corpus callosum and CSO compared to HIV− controls and HIV+/cART+ individuals. No significant difference existed between HIV− controls and HIV+/cART+ patients. cART initiation significantly improved mean diffusivity, radial diffusivity, and axial diffusivity, but not fractional anisotropy, in the corpus callosum and CSO in some HIV-infected patients.Observed decreases in DTI parameters between HIV+/cART+ and HIV+/cART− individuals could reflect the presence of inflammatory cells or cytotoxic edema in HIV+/cART− patients. Initiating cART could lead to a reduction in neuro-inflammation and improvement in DTI measures. Future DTI studies may be useful for evaluating the efficacy higher brain penetrating cART regimens.