The potential molecular mechanism of apoptosis of mouse spermatogonial GC-1 cells under hy-poxia was investigated. First, the cell viability under hypoxia for different time periods was measured using the Cell Counting Kit-8 (CCK-8) to determine the time of cell damage induced by hypoxia. Then, the content of reactive oxygen species (ROS) in GC-1 cells was detected by chemical fluorescence method, the membrane potential of mitochondria was detected by JC-1 assay, and the content of ATP was detected by colorimetry. Meanwhile, the number and distribution of mitochondria were detected using fluorescent probe method, and the ultrastructure of the cells was observed by transmission electron microscopy. Finally, mRNA expression levels of mitochondrial signaling pathway related genes caspase-3, caspase-9, cytochrome c (Cyt-c), Bax and Bcl-2 were analysed using real-time fluorescence quantitative PCR. The results showed that, after 36 h of hypoxia, the cell viability was (60.36±5.40)%, which met the requirement of subsequent experiments. Under hypoxia conditions, the ROS content in GC-1 cells increased significantly, and the ATP content decreased significantly. The membrane potential and number of mitochondria were reduced. At the same time, the ex-pression levels of pro-apoptosis-related genes were up-regulated, and the gene expression level of anti-apoptotic factor Bcl-2 was down-regulated. The results showed that hypoxia can lead to mitochondrial dys-function in GC-1 cells, and ROS/mitochondrial signaling pathway may be the molecular mechanism of hy-poxia-induced apoptosis of GC-1 cells.