Spinal cord injury (SCI) is a devastating trauma that results in a severe disability and irreversible motor and sensory dysfunction, whereas efficient therapies have not been fully developed. After SCI, astrocytes are the predominant cellular component that proliferates around the lesion core and contributes to the glial scar formation, which has long been considered one of the primary causes of spinal cord regeneration failure. However, the molecular mechanisms underlying the proliferation of astrocytes in response to central nervous system (CNS) injury remain unclear. In this study, we found that heterogeneous nuclear ribonucleoprotein U (Hnrnpu), a DNA/RNA binding protein, regulated astrocyte proliferation after SCI. siRNA-mediated knockdown of Hnrnpu suppressed the primary astrocyte proliferation without affecting the cell viability in vitro. Moreover, in vivo, inhibition of Hnrnpu expression by intraspinal injection of AAV5-Hnrnpu shRNA under the control of the astrocytic glial fibrillary acidic protein (GFAP) promoter inhibited astrocyte proliferation, increased lesion size, and suppressed functional recovery of mice after SCI. Taken together, Hnrnpu exerts a crucial role in astrocyte proliferation, where its changes would be regarded as a hallmark of CNS diseases and injuries in which astrocytes are involved.