Diffusion-weighted imaging (DWI) measures brain water diffusion that is sensitive to microscopic brain injury. A total of 11 HIV seropositive patients were compared to 14 seronegative subjects using DWI, proton magnetic resonance spectroscopy (1H MRS), and neuropsychological tests. The apparent diffusion coefficient (ADC) was significantly increased in the HIV patients, primarily in the frontal white matter (FWM; +5%, p=0.01). Diffusivity correlated positively with the glial marker myo-inositol (r=0.5, p=0.008) and negatively with cognitive performance (NPZ-8 composite score; r=-0.43, p=0.05). These findings suggest increased brain water diffusion may reflect increased glial activation or inflammation, which in turn, may contribute to the cognitive deficits in HIV patients.