The major protein of horse seminal plasma, HSP-1/2, exhibits membranolytic and chaperone-like activities and plays acrucial role in regulating sperm capacitation. L-Carnitine is a small polar molecule present in high concentrations inmammalian seminal plasma. The present results demonstrate that L-carnitine binds to HSP-1/2 and increases its thermalstability, enhances cooperativity of its chemical unfolding and decreases both chaperone-like and membranolytic activitiesof this protein. The HSP-1/2–L-carnitine complex exhibits anti-oxidative behaviour by inhibiting the production ofhydroxyl radicals, suggesting that it can protect other constituents of seminal plasma from damage by hydroxyl radicals. AsHSP-1/2 and L-carnitine share the same spatiotemporal location in the horse reproductive tract, this interaction is physiologicallysignificant and may prevent premature interaction of HSP-1/2 with sperm, which in turn regulates the spermcapacitation.