Cerebral small vessel disease results in lacunar infarcts and cognitive impairment. Diffusion tensor imaging (DTI) demonstrates a reduction in fractional anisotropy and increase in mean diffusivity, which correlates more strongly with cognition than conventional MRI. The underlying pathological basis for these DTI changes is not known. In this study magnetic resonance spectroscopy was used to determine the biochemical basis of these DTI alterations. Twenty-five patients with lacunar stroke and radiological leukoaraiosis were recruited. Chemical shift imaging (CSI) and DTI were performed on a 1.5 T MRI scanner. A region of interest was positioned in the white matter of the centrum semiovale. Multivoxel CSI data were processed and the metabolite ratios estimated. DTI parameters corresponding to the exact region of tissue excited by CSI were obtained. Mean spectroscopy data and DTI values for each subject were correlated. Univariate analysis revealed a positive correlation between N-acetyl aspartate-creatine (NAA/Cr) and fractional anisotropy (r = 0.52, p = 0.008), and a negative correlation with mean diffusivity (r = -0.51, p = 0.009). Results remained little changed after controlling for mean percentage lesion and mean percentage white matter per voxel (with fractional anisotropy r = 0.54, p = 0.008, and with mean diffusivity r = -0.52, p = 0.01). These findings are consistent with axonal loss or dysfunction, or both, accounting for at least part of the DTI abnormalities found in patients with small vessel disease. It provides evidence that DTI identifies axonal disruption in white matter tracts.