Background: Previous reports suggested that methamphetamine (METH) exposure could lead to inhibition of rat testis spermatogenesis. Glycolysis and glucose metabolism as well as oxidative stress have been implicated in testis spermatogenesis. Here we explored the underlying mechanism of local metabolism and glycolysis of testis after METH exposure. Material/Methods: METH was intraperitoneally injected into rats with different doses and duration of METH exposure to establish short-term and chronic exposure models. The serum 8-hydroxy-2 deoxyguanosine (8-OHdG) level of rats was detected by enzyme-linked immunosorbent assay. Untargeted gas chromatography-mass spectrometry analysis was applied to identify differential metabolites and metabolic signature. The mRNA expression of hypoxia inducible factor 1α (HIF1α), glucose transporter 1 (GLUT1), hexokinase 1 (HK1) and lactate dehydrogenase C (LDHC) in rat testes were detected by polymerase chain reaction. Further, we determined the 4 proteins with western blotting and immunohistochemistry. Results: Decreased testes index and sperm counts were showed in the chronic METH group. The metabolome revealed that the main differential metabolites impacted were associated with glycolysis and glucose metabolism. The mRNA and protein expression of GLUT1, HK1, and LDHC were reduced in the chronic METH group but elevated in the short-term METH group, whereas HIF1α was upregulated in the short-term METH group but remained at baseline in the chronic METH group. Conclusions: Overall, glucose metabolism was regulated by HIF1a after short-term METH exposure. Reduced glycolysis in the testis led to impaired spermatogenesis after chronic METH exposure. © Med Sci Monit.