LEV improves the percentage of forward-motion spermatozoon and total sperm motility in patients with oligozoospermia or asthenospermia in clinical settings. However, the mechanism of action of levocarnitine (LEV) in the treatment of spermatogenic dysfunction was unclear. Based on in vitro and in vivo experiments, we used Glucosides of Tripterygium wilfordii Hook F (GTW) to construct a cell model (using spermatogenic GC-1 spg cells) and an animal model (using rats) of spermatogenic dysfunction. LEV and LY294002 (a PI3K pathway inhibitor) were then administered. By assessing apoptosis and sperm quality and motility, the underlying mechanism was explored. We found that GTW induced spermatogenic dysfunction, and LEV ameliorated the GTW-induced spermatogenic dysfunction. LEV inhibited GC-1 spg cell apoptosis and improved the sperm count and percentages of PR (forward motion) + NP (non-forward motion) (p < .01). Besides, the morphology of testicular tissue in the GTW + LEV and LY + LEV groups was superior to that in the GTW group. We can to the conclusion that LEV may operate via the PI3K/AKT signalling pathway, with increases in PI3K, p-AKT, and BCL-2 protein and mRNA expression, so that the percentages of GC-1 spg cells apoptosis decrease, and the sperm count and motility improve.