Summary: NEUROD1 (ND1)-induced astrocyte-to-neuron (AtN) conversion shows promise for treating neurological disorders. To gain insight into the molecular mechanisms of neuronal reprogramming, we established an in vitro system using primary cortical astrocyte cultures from postnatal rats and employed single-cell and multiomics sequencing. Our findings indicate that the initial cultures primarily consisted of immature astrocytes (ImAs), with potentially a minor presence of radial glial cells. The ImAs initially went through an intermediate state, activating both astrocyte and neural progenitor genes. Subsequently, they mimic in vivo neurogenesis to acquire mature neuronal characteristics. We show that ND1 acted as a pioneer factor that reshapes the chromatin landscape of astrocytes to that of neurons. This restructuring promotes the expression of neurogenic genes via inducing H3K27ac modification. Through integrative analysis of various ND1-induced neuronal specification systems, we identified 25 ND1 targets, including Hes6, as key regulators. Thus, our work highlights the key role of ND1 and its downstream regulators in neuronal reprogramming.