Most gastropods deposit their eggs either on land or underwater, but the family Ampullariidae includes members that exhibit both underwater and aerial oviposition, making it an ideal model for understanding mechanisms underlying the evolutionary transition from water to land. We applied SDS-PAGE and LC-MS/MS to analyse the proteome of the egg perivitelline fluid (PVF) of Marisa cornuarietis – an aquatic ovipositing ampullariid. Comparison with the reported PVF proteomes of two aerial ovipositing ampullariids (Pomacea canaliculata and P. maculata) showed that the three species all contain several major perivitellins that nourish the embryos. However, M. cornuarietis invests more heavily on immune-related proteins, which might be due to exposure to aquatic pathogens. Interestingly, only the PVF of out-of-water egg laying species have PV2 – a neurotoxin lethal to mice, and a calcium-binding protein which might be involved in the formation of calcareous eggshell. Integrated phylogenetic, evolutionary and gene expressional analyses detected the involvement of gene duplication, positive selection and neofunctionalisation in the formation of several major PVF proteins. Overall, our study provides multiple lines of evidence of adaptive evolution in the PVF proteins, and contributes to a better understanding of how aquatic gastropod ancestors invaded terrestrial habitats.