5-Fluorouracil (5-FU) is a widely used anticancer drug that acts by blocking DNA replication. Although the side effects of 5-FU are well documented, the mechanism of 5-FU-induced neurotoxicity remains unclear. The current study was performed to investigate the toxicity of 5-FU to rat cerebellum granule cells (CGCs) and to elucidate the corresponding molecular mechanisms. We demonstrated that 5-FU exhibited significant cellular toxicity to CGCs, in a dose-dependent manner, and that it altered intracellular Ca2+ levels. The accumulation of intracellular reactive oxygen species in CGCs revealed that 5-FU also induced oxidative stress. Moreover, 5-FU-treated cells showed elevated caspase-3 activity. Furthermore, intraperitoneally administered 5-FU caused slight degenerative changes in the rat cerebellum granular layer. Taken together, these findings revealed that 5-FU substantially impaired the survival of CGCs by inducing oxidative stress and activating the caspase-3 apoptotic pathway.