〔Abstract〕 To explore the effects of hypoxia on the migration ability of human oligodendrocyte precursor cells (hOPCs) and its regulatory mechanisms. Methods Based on the variations in oxygen concentration within the culture system, three experimental groups were set up: the 21% O₂ group (normoxic control group), the 5% O₂ group, and the 2% O₂ group. The migration ability of hOPCs under normoxia (21% O₂), 5% O₂, and 2% O₂ conditions was detected through the Transwell migration assay. RT-qPCR, transcriptome sequencing, and flow cytometry were used to detect the expression changes of genes and proteins such as hypoxia-inducible factor 1 alpha (HIF-1α) and Chemokine (C-X-C Motif) Receptor 4 (CXCR4). Bioinformatics analysis was combined to analyze the KEGG pathways related to migration, so as to explore the effects of different oxygen concentrations on the migration ability of hOPCs and their possible mechanisms. Results Hypoxia treatments at concentrations of 5% O₂ and 2% O₂ could both promote the in vitro migration of hOPCs, and the promoting effect of migration was more significant at the 2% O₂ concentration (P<0.001). After hypoxia treatment, the mRNA expression levels of HIF-1α, CXCR4, etc. in hOPCs significantly increased (P<0.001). Compared with the 5% O₂ concentration, the expression of CXCR4 in cells was higher at the 2% O₂ concentration (P<0.000 1). Flow cytometry analysis detection showed that the expression of CXCR4 increased significantly after hypoxia treatment (P<0.01), and with the decrease of oxygen concentration, its expression level further increased (P<0.000 1). Ordinary transcriptome sequencing analysis indicated that hypoxia treatment could activate the PI3K-Akt signaling pathway and the Axon guidance pathway. Conclusion In conclusion, hypoxia treatment can enhance the in vitro migration ability of hOPCs, and this effect is negatively correlated with the oxygen concentration. Its mechanism may be related to the up-regulation of the expression of genes such as HIF-1α and CXCR4, and the activation of the migration related signaling pathway including PI3K-Akt signaling pathway and Axon guidance pathway.