As a highly plastic population, parenchymal astrocytes have demonstrated the capacity to become activated and recapitulate neurogenic potential in response to traumatic central nervous system (CNS) injuries, representing a latent reservoir for neuronal regeneration in non-neurogenic brain regions. However, the extrinsic and intrinsic factors regulating this process remain poorly characterized. Elucidating these molecular mechanisms is crucial to harnessing the regenerative potential of reactive astrocytes in CNS repair. A multidisciplinary approach combining immunostaining, western blotting, RNA interference (RNAi), gene knock out and fate-mapping was used to investigate the role of topoisomerase IIα (TOP2a) in regulation of the stemness response in reactive astrocytes to traumatic brain injury (TBI). Both and analyses demonstrated that TBI induces a stem cell-like response in reactive astrocytes concomitant with TOP2a upregulation. Pharmacological inhibition or genetic deletion of TOP2a significantly attenuated this stemness response. Neurosphere culture assay indicates that TOP2a might act as a downstream factor of Sonic Hedgehog (SHH) signaling to mediate the acquisition of stem cell potential. This study identifies TOP2a as a pivotal intrinsic regulator of astrocytic stem cell potential in the injured brain, which will advance our understanding of the molecular underpinnings of the stem cell response and its therapeutic application in neural regeneration.