Astrocytes communicate bidirectionally with neurons by forming close contacts to synapses called tripartite synapses. Hundreds of thousands of synapses are embedded in a territory of single astrocytes, however, how astrocytes are functionally built into the neuronal circuits remains undetermined. To address this, we adopted the rabies virus (RV) for retrograde tracing of neuron-to-astrocyte connections in the mouse cerebral cortex. To sparsely and specifically transduce astrocytes with RV, a transposon vector expressing TVA, RV glycoprotein, and tdTomato under an astrocyte-specific promoter Gfa2 was introduced by electroporation at postnatal day (P) 0. Then, EnvA-pseudotyped and glycoprotein-deleted RV (EnvA-RV∆G-GFP) was injected at P30–50, and the brains were collected after five days of transduction. Neurons expressing only GFP were found around astrocytes expressing both tdTomato and GFP, demonstrating that EnvA-RV∆G-GFP primarily infected astrocytes and then spread to neurons. Secondarily infected neurons were also found in distant cortical regions, indicating that astrocytes receive long-range cortico-cortical projections as well as local connections. Topological analysis further revealed layer-specific neuron-to-astrocyte projections: layer I astrocytes receive horizontally widespread projection from infragranular layers, layer II/III astrocytes receive columnar inputs, and layers V and VI astrocytes mainly receive interlaminar projections. Moreover, astrocytic calcium response patterns evoked by local electrical stimulations to neurons coincided with the RV-revealed wiring diagram. Taken together, RV tracing enabled intra- and inter-regional mapping of neuron-to-astrocyte connections, thus is a potential tool for dissecting the astrocyte-neuron circuit organization.