Rabies virus (RABV) causes severe rabies, which still poses a great threat to human and animal health worldwide due to its almost 100% mortality rate. There is no effective therapy for this lethal disease. A better understanding of the mechanism of RABV infection is important to develop effective therapies. RABV M protein plays multiple roles in RABV infection; however, its function during the early phase of viral infection remains unknown. To explore the role of M protein in RABV infection of host cells, we identified host proteins that interacted with the M proteins by using mass spectrometry. In this study, we discovered that ATP6V1A interacts with RABV M protein, and facilitates RABV replication by promoting viral uncoating. Our results demonstrate that RABV M protein plays important roles in the early stage of the RABV life cycle. In addition, stably transfected Vero cells overexpressing ATP6V1A promoted the propagation of RABV, which could be a strategy to improve rabies virus vaccine production.