It has been recently shown in mice that sperm undergo acrosome reaction (AR) by passing through cumulus cells; furthermore, the acrosome-reacted sperm can bind to zona pellucida and consequently fertilise the egg. During AR, the relocation of the primary fusion protein IZUMO1 into the equatorial segment is crucial for sperm-egg fusion. There is a high rate of spontaneous AR in rodents, with up to 60% in promiscuous species. The aim of this study was to clarify whether the IZUMO1 relocation in sperm after spontaneous and induced AR is the same, and whether there is a correlation between the speed of IZUMO1 relocation and species-specific mating behaviour in field mice. Immunofluorescent detection of IZUMO1 dynamics during the in vitro capacitation, spontaneous, calcium ionophore and progesterone-induced AR was monitored. Our results show that during spontaneous AR, there is a clear IZUMO1 relocation from the acrosomal cap to the equatorial segment, and further over the whole sperm head. In addition, there is positive tail tyrosine phosphorylation (TyrP) associated with hyperactive motility. Moreover, the beginning and the progress of IZUMO1 relocation and tail TyrP positively correlate with the level of promiscuity and the acrosome instability in promiscuous species. The findings that crucial molecular changes essential for sperm-egg fusion represented by dynamic movements of IZUMO1 also happen during spontaneous AR are vital for understanding fertilisation in mice.