The protein hormone prolactin (PRL) was first discovered as an anterior pituitary factor capable of stimulating milk production in mammals. We now know that PRL has over 300 different functions in vertebrates. In fish, PRL plays an important role in freshwater osmoregulation by preventing both the loss of ions and the uptake of water. This paper will review what is currently known about the structure and evolution of fish PRL and its mechanisms of action in relation to the maintenance of hydromineral balance. Historically, functional studies of fish PRL were carried out using heterologous PRLs and the results varied greatly between experiments and species. In some cases this variability was due to the ability of these PRLs to bind to both growth hormone and PRL receptors. In fact, a recurring theme in the literature is that the actions of PRL cannot be generalized to all fish due to marked differences between species. Many of the effects of PRL on hydromineral balance are specific to euryhaline fish, which is appropriate given that they frequently experience sudden changes in environmental salinity. Much of the recent work has focused on the isolation and characterization of fish PRLs and their receptors. These studies have provided the necessary tools to obtain a better understanding of the evolution of PRL and its role in osmoregulation.