Meditation has a versatile nature to affect cognitive functioning of human brain. Recent researches demonstrated its effects on white matter (WM) properties of human brain. In this research, we aim to investigate WM microstructure of corpus callosum (CC) in long-term meditators (LTMs) of rajayoga meditation using diffusion tensor imaging. For this cross-sectional analysis, 22 LTMs and 17 control participants of age ranging from 30 to 50 years were recruited. Results show high fractional anisotropy values with low mean diffusivity in whole as well as different segments of CC in the LTM group. Also the experience of meditation was correlated with WM properties of CC tracts. Findings may suggest rajayoga meditation to bring potential changes in microstructure of CC segments. Further studies are suggested in clinical population to check its validity and efficacy against disorders involving agenesis of WM.