Objective: Fluoride increases the production of reactive oxygen and nitrogen derivatives, causing oxidative stress and cellular damage. Betaine, an antioxidant and an important methyl donor, has been reported to have potential protective effects on cardiovascular and neurodegenerative diseases in recent years. In this study, we aimed to investigate the neuroprotective effects of betaine treatment against cellular damage caused by fluoride exposure on rat synaptosomes. Material and Methods: In the experimental period, 8 Wistar albino rats were decapitated and then their frontal cortices removed and divided into 4 equal cuts (total-32 cuts). Subsequently, an appropriate experimental procedure was applied to obtain the synaptosomal fractions. The determined doses of sodium fluoride (NaF) and betaine were administered in vitro at 37°C for 30 min. Synaptosomal glutathione (GSH), malondialdehyde (MDA) and nitricoxide (NO) levels, and also catalase (CAT) and Ca+2/Mg+2 ATPase activities were measured spectrophotometrically. Results: According to our results, NaF exposure caused a decrease in GSH levels, CAT and Ca+2/Mg+2 ATPase activities, and also an increase in MDA and NO levels, significantly. MDA levels, NO levels and CAT activities were closed to the control group depends on the betain doses. The best recovery in terms of synaptosomal GSH levels and Ca+2/Mg+2 ATPase activities was found at 0.5 mM betaine concentration (P <0.001). Conclusion: Our results showed that betaine could be a potential neuroprotective therapeutic agent against synaptosomal fluoride toxicity.