The plateau environment impacts male reproductive function, causing decreased sperm quality and testosterone levels.l-carnitine can improve the semen microenvironment. However, the role ofl-carnitine in a high-altitude environment remains unclear. In our study, we investigated the effects ofl-carnitine administration in a male Wistar rat reproductive system injury model in the context of a simulated high-altitude environment. Rats were randomly divided into a normal control group (group A1, A2-low dose and A3-high dose) and high-altitude model groups (group B, C-low dose and D-high dose) with 20 rats in each group. With the exception of the normal control group exposed to normoxic conditions, the other groups were maintained in a hypobaric oxygen chamber that simulated an altitude of 6000 m for 28 days. In the experimental period, the low-dose groups (A2 and C) were administered 50 mg/kgl-carnitine via intraperitoneal injection once a day, and the high-dose groups (A3 and D) were given 100 mg/kg. After the feeding period, blood samples were collected to assess blood gas, serum hormone levels and oxidative stress. Sperm from the epididymis were collected to analyse various sperm parameters. After obtaining the testicular tissue, the morphological and pathological changes were observed under a light microscope and transmission electron microscopy (TEM). The impact of the simulated high-altitude environment on the rat testis tissue is obvious. Specifically, a decreased testicular organ index and altered indices of arterial blood gas and serum sex hormone levels caused testicular tissue morphological damage, reduced sperm quality, increased sperm deformity rate and altered malondialdehyde (MDA), superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) concentrations. The results demonstrate thatl-carnitine can be administered as a preventive intervention to reduce the reproductive damage caused by high-altitude hypobaric and hypoxic environments and improve semen quality in a rat model.