Mindfulness-based stress reduction (MBSR) has been known to improve emotion regulation and cure mood disorders. The present longitudinal study aimed to clarify the effects of MBSR training and practice on the neural substrates of emotion regulation. Resting-state functional MRI (rsfMRI) exams were performed at three time points to investigate changes of functional connectivity (FC) within the subgenual anterior cingulate cortex (sgACC) network. MBSR training and practice strengthened the FC of sgACC with the inferior frontal gyrus and with the insula, and reduced the level of anxiety and depression. The findings reveal the neural substrates of emotion regulation process following MBSR.