The vomeronasal organ (VNO) is a chemosensory organ present in most vertebrates and involved in chemical communication. Last decades, the deciphering of the signal transduction process of this organ has progressed. However, less is known about the VNO ligands and their structure-function relationships. Snakes possess a highly developed vomeronasal system which is used in various behaviours such as mating, predator detection or prey selection, making this group a suitable model for study of the vomeronasal chemoreception. In this work, we used a proteomic approach to identify and characterize proteins from frog cutaneous mucus proteome involved in prey recognition by snakes of the genus Thamnophis. Herein we report the first purification and characterization of two proteins isolated from the frog skin secretome that elicit the vomeronasal-mediated predatory behaviour of Thamnophis marcianus. These proteins are members of the parvalbumin family which are calcium- binding proteins generally associated to muscular and nervous tissues. This is the first report that demonstrates parvalbumins are not strictly restricted to intracellular compartments and can also be isolated from exocrine secretions. Purified parvalbumins from frog’s muscle and mucus revealed identical chemoattractive properties for Thamnophis marcianus. Snake bioassay reveals Ca²+/Mg²+ dependence of the parvalbumins’ bioactivity. So, parvalbumins appear as new candidate ligands of the vomeronasal organ.