Objectives Sequelae of stroke were mainly caused by neuronal injury. Oxygen is a key factor affecting the microenvironment of neural stem cells (NSCs), and oxygen levels are used to promote NSC neurogenesis. In this study, effects of intermittent hypoxic preconditioning (HPC) on neurogenesis were investigated in a rat model of middle cerebral artery occlusion (MCAO). Methods SD rats were used to establish the MCAO model. Nissl staining and Golgi staining were used to confirm the neuronal injury status in the MCAO model. Immunofluorescence, transmission electron microscopy, Western blot, and qPCR were used to observe the effects of HPC on neurogenesis. At the same time, the hypothesis that HPC could affect proliferation, apoptosis, differentiation, and migration of NSC was verified in vitro. Results Hypoxic preconditioning significantly ameliorated the neuronal injury induced by MCAO. Compared with MCAO group, the dendrites, Edu+/SOX2+, Edu+/DCX+, Edu+/NeuN+, Edu+/GFAP+, and Edu+/Tubulin+ positive cells in the HPC + MCAO group exhibited significantly difference. Similarly, axonal and other neuronal injuries in the HPC + MCAO group were also ameliorated. In the in vitro experiments, mild HPC significantly enhanced the viability of NSCs, promoted the migration of differentiated cells, and reduced apoptosis. Conclusions Our results showed that HPC significantly promotes neurogenesis after MCAO and ameliorates neuronal injury. 1. HPC alleviated the nerve injury of MCAO; 2. HPC promoted neurogenesis; 3. HPC affects proliferation and apoptosis of NSCs.