White matter (WM) deterioration is an important mechanism of cognitive decline in healthy aging and dementia. Engaging in aerobic exercise to improve cardiorespiratory fitness (CRF) is considered one of the most promising ways to improve cognitive and brain health in aging. Yet, no randomized controlled trials have reported benefits of aerobic exercise interventions on WM microstructure measured with diffusion tensor imaging. Here, we studied the effects of a 6-month exercise intervention (clinical trial NCT01472744) on WM of 180 cognitively healthy older adults (60–79 years) using the ratio of calibrated T1-weighted image to T2-weighted images (T1w/T2w). Participants were randomized to one of four groups including a low intensity activity with complex cognitive demands (Dance), Walking, Walking + nutritional supplement or an active control. Results showed that a 6-month aerobic walking and dance intervention produced positive changes in the T1w/T2w with significant time-by-group interactions in the total WM, the genu and splenium of the corpus callosum, forceps minor, cingulum, relative to an active control condition. In contrast, we observed a decline in T1w/T2w in the majority of WM regions in the active control group. Lastly, a positive change in the T1w/T2w in the genu of the corpus callosum correlated with a positive change in episodic memory in the Walking but not in the control group; however, there were no associations between change in the T1w/T2w and change in CRF. Together, our findings suggest that the T1w/T2w may be a sensitive metric to detect short-term within-person changes in the WM and intervention-induced WM plasticity in the adult human brain.