Abstract: To investigate the effects of acetyl-L-carnitine（ALC） on mitochondrial respiratory function and autophagy in mouse spermatogonia cell line under hypoxia, GC-1 spermatogonia cell line was used as the research object, which was devided into 3 groups: control group（5%CO₂ and 95%AIR）, hypoxia group（5%CO₂ and 3%O₂） and hypoxia+ALC group（5%CO₂ and 3%O₂, the medium contained with 150 mmol/L ALC）. After treatment for 24 hours, the changes of related indexes of mitochondrial respiratory function were detected by high-resolution respirator. The mRNA relative expression levels of autophagy related-genes were detected by qRT-PCR, and the expression levels of autophagy related proteins were detected by Western-blot. The results showed that compared with control group, the basal respiration, proton leak, ATP-linked respiration, maximal respiration, spare respiratory capacity and respiratory control rate were extremely significantly decreased（P＜0.01）, while the residual oxygen consumption increased slightly in hypoxia group（P＞0.05）. However, ALC supplementation did not significantly improve the changes of above indexes induced by hypoxia（P＞0.05）. Compared with control group, the mRNA relative expression levels of ATG5, Beclin1, p62 genes in hypoxia group were extremely significantly down-regulated（P＜0.01）, the mRNA relative expression levels of LC3 b were slightly up-regulated but the difference was not significant（P＞0.05）, and the expression of autophagy related protein Beclin1, ATG5, LC3 b/LC3 a down-regulated significantly（P＜0.05）. Compared with hypoxia group, the supplementation of ALC significantly or very significantly reversed the down-regulation of mRNA expression of autophagy related genes ATG5, Beclin1, p62（P＜0.05 or P＜0.01）, while the supplementation of ALC significantly up-regulated the relative expression levels of LC3 b（P＜0.05）. The relative expression levels of LC3 b/LC3 a protein were extremely significantly up-regulated（P＜0.01）, while the relative expression levels of ATG5 and Beclin1 protein were up-regulated but there was no significant difference（P＞0.05）. The results indicated that ALC might activate the autophagy inhibition of GC-1 spermatogonia induced in hypoxia environment, but it could not reverse mitochondrial respiratory dysfunction.