It was reported that CXCR4 signaling played an important role in the migration and differentiation of endogenous neural stem cells after spinal cord injury (SCI). However, the molecular mechanism of it is still unclear. Here, we established a model of SCI in rats and AMD3100 was used to treat them. The rats were then sacrificed and the injured spinal cord specimens were harvested. Additionally, the neural stem cells (NSCs) line was culture and treated with AMD3100 in vitro. Results showed the locomotor function of SCI rats was worse after treated with AMD3100. And the expression levels of Nestion in neural stem cells and β-tubulin in neuron cells were significantly increased in the injured spinal cord, which can be inhibited by the CXCR4 antagonist of AMD3100. Additionally, the expression of β-catenin and phosphorylase β-catenin protein was significantly down regulated by AMD3100. In vitro, the NSCs proliferation ability was inhibited and the migration was decreased after treated with AMD3100. Also, the expression of Nestion, β-tubulin, β-catenin and phosphorylase β-catenin protein was significantly decreased in AMD3100 group comparing with untreated group. Taken together, this study suggested that AMD3100 could inhibit the migration and differentiation of endogenous neural stem cells in rats with SCI. The mechanism of it maybe that AMD3100 could down regulate of SDF-1/CXCR4 by targeting β-catenin signaling pathway.