Fluoride is a widespread environmental pollutant which can induce low sperm quality and fertilizing ability, however, the underlying mechanism is still remaining unclear. Hence, we aimed to investigate the influence of fluoride on sperm fertilizing ability via some key proteins in epididymis. For this, forty adult rats were assigned randomly into four groups. The control group was given distilled water, while the other three groups were given with 25, 50, and 100mg NaF/L via drinking water for 56 d, respectively. Post one day, epididymides were processed for sperm-egg binding, RNA extraction, western blot and immunofluorescence analysis. Fluoride exposure reduced the ability of sperm to break down the egg cumulus cell layer. Further study revealed that fluoride altered the expression levels of genes and proteins related to acrosome reaction in vivo, including SPAM1, ACR, and PRSS21. However, fluoride only affected the expression of ACR protein only in the epididymis, but not in the testis. Fluoride also affected the expression levels of the membrane proteins CD9 and CD81 of epididymosomes in the epididymis. From the results, it can be concluded that fluoride exposure reduced the ability of sperm to break down the egg cumulus cell layer, which could be one of the reason for decreased fertility ability in males treated with Fluoride.. These results provide some theoretical guidance and new ideas for treatments of low fertility, infertility and other reproductive diseases.