It is well established that mammalian spermatozoa acquire functional maturity during two key phases of post-testicular development. The first of these occurs as the cells are conveyed through the male reproductive tract (epididymal maturation) and the second as they ascend the female reproductive tract (capacitation). However, the degree of post-testicular development necessary to achieve fertilisation in other vertebrates remains far less clear. Indeed, despite reports that the Wolffian duct of aves and reptiles is capable of secreting proteins that bind and modify the sperm surface characteristics, it remains contentious whether capacitation is a pre-requisite for fertilisation in these species. This study was therefore undertaken with the aim of exploring whether reptile sperm do undergo capacitation-like changes following ejaculation. For this purpose we assessed the behaviour of Australian saltwater crocodile (Crocodylus porosus) spermatozoa in response to incubation under conditions that have been optimised for the induction of capacitation in mammalian species. These studies revealed that crocodile spermatozoa experienced a rapid, cyclic-AMP mediated increase in progressive motility that was accompanied by elevated levels of tyrosine phosphorylation predominantly localised within the sperm tail. These characteristic hallmarks of capacitation were also accompanied by a concomitant increase in the capacity to undergo an ionophore induced acrosome reaction. Among the key targets for tyrosine phosphorylation, we identified an orthologue of outer dense fibre 2 (ODF2), a protein that also undergoes capacitation-associated tyrosine phosphorylation in mammalian spermatozoa. Overall these data support the concept that reptilian spermatozoa do undergo capacitation-like changes in preparation for fertilisation of an ovum. Such findings are likely to find application in the development of innovative assisted reproductive strategies for captive populations of crocodiles.