In vivo induction of non-neuronal cells into neurons by transcription factors offers potential therapeutic approaches for neural regeneration. Although generation of induced neuronal (iN) cells in vitro and in vivo has been reported, whether iN cells can be fully integrated into existing circuits remains unclear. Here we show that expression of achaete-scute complex homolog-like 1 (Ascl1) alone is sufficient to convert dorsal midbrain astrocytes of mice into functional iN cells in vitro and in vivo. Specific expression of Ascl1 in astrocytes by infection with GFAP-adeno-associated virus (AAV) vector converts astrocytes in dorsal midbrain, striatum, and somatosensory cortex of postnatal and adult mice into functional neurons in vivo. These iN cells mature progressively, exhibiting neuronal morphology and markers, action potentials, and synaptic inputs from and output to existing neurons. Thus, a single transcription factor, Ascl1, is sufficient to convert brain astrocytes into functional neurons, and GFAP-AAV is an efficient vector for generating iN cells from astrocytes in vivo.