While the swiftly evolving technique of in vitro fertilisation has been widely successful in other domesticated species, the development of a robust system of IVF for the horse remains an elusive and highly valued goal. This problem is, in part, attributed to a concerning inability to produce populations of capacitated stallion spermatozoa that are competent to engage in oocyte interactions. The basic conditions employed to capacitate stallion sperm have thus far failed to elicit the full spectrum of functional endpoints associated with this process and these studies have been further limited by their reliance upon the cholesterol sequestering action of animal-derived serum albumin. To address these concerns this study evaluated markers of sperm function following incubation in media optimised to promote in vitro capacitation. This study revealed for the first time that robust in vitro capacitation of stallion sperm can be achieved in a bicarbonate rich media supplemented with a phosphodiesterase inhibitor (pentoxifylline), a cAMP analogue (dibutyryl cAMP), and an efficient cholesterol withdrawing agent (methyl-β-cyclodextrin) other than serum albumin. The populations of spermatozoa generated under these conditions displayed a number of hallmarks of capacitation, including: elevated levels of tyrosine phosphorylation, a significant increase in hyperactivated motility (p<0.05), and a reorganisation of the plasma membrane leading to lipid raft coalescence at the apical region of the sperm head. Importantly, these changes were positively correlated with a dramatic increase in the ability of these cells to interact with heterologous bovine ZP (p<0.001) and undergo agonist induced acrosomal exocytosis (p<0.05). Collectively these results suggest that highly efficient cholesterol removal from the stallion sperm plasma membrane may be essential to permit the movement of lipid rafts to the apical region of the sperm head which may be an essential priming event for ZP binding and the subsequent fertilisation of oocytes in vitro.