Fluoride is known to cause male reproductive toxicity, and the elucidation of its underlying mechanisms is an ongoing research focus in reproductive toxicology and epidemiology. Choline, an essential nutrient, has been extensively studied for its benefits in nervous system yet was rarely discussed for its prospective effect in male reproductive system. This study aims to explore the potential protective role of choline against NaF-induced male reproductive toxicity via MAPK pathway. The male mice were administrated by 150 mg/L NaF in drinking water, 5.75 g/kg choline in diet, and their combination respectively from maternal gestation to postnatal 15 weeks. The results showed that fluoride exposure reduced body weight growth, lowered sperm count and survival percentages, altered testicular histology, down-regulated the mRNA expressions of NGF, Ras, Raf, and MEK genes in testes, as well as significantly decreased the expressions of both NGF and phosphor-MEK proteins in testes. Examination of data from choline-treated mice revealed that choline supplementation ameliorated these fluoride-induced changes. Taken together, our findings suggest that choline supplementation alleviates fluoride-induced testicular toxicity by restoring the NGF and phosphor-MEK expression. The suitable dosage and supplementation periods of choline await further exploration.