A new technology called in vivo glia-to-neuron conversion has emerged in recent years as a promising next generation therapy for neural regeneration and repair. This is achieved through reprogramming endogenous glial cells into neurons in the central nervous system through ectopically expressing neural transcriptional factors in glial cells. Previous studies have been focusing on glial cells in the grey matter such as the cortex and striatum, but whether glial cells in the white matter can be reprogrammed or not is unknown. To address this fundamental question, we express NeuroD1 in the astrocytes of both grey matter (cortex and striatum) and white matter (corpus callosum) to investigate the conversion effciency, neuronal subtypes, and electrophysiological features of the converted neurons. We discover that NeuroD1 can effciently reprogram the astrocytes in the grey matter into functional neurons, but the astrocytes in the white matter are much resistant to neuronal reprogramming. The converted neurons from cortical and striatal astrocytes are composed of both glutamatergic and GABAergic neurons, capable of firing action potentials and having spontaneous synaptic activities. In contrast, the few astrocyte-converted neurons in the white matter are rather immature with rare synaptic events. These results provide novel insights into the differential reprogramming capability between the astrocytes in the grey matter versus the white matter, and highlight the impact of regional astrocytes as well as microenvironment on the outcome of glia-to-neuron conversion. Since human brain has large volume of white matter, this study will provide important guidance for future development of in vivo glia-to-neuron conversion technology into potential clinical therapies. Experimental protocols in this study were approved by the Laboratory Animal Ethics Committee of Jinan University (approval No. IACUC-20180321-03) on March 21, 2018.