Synaptotagmins (Syt) are a large family of proteins that regulate membrane traffic in neurons and other cell types. One isoform that has received considerable attention is SYT4, with apparently contradictory reports concerning the function of this isoform in fruit flies and mice. SYT4 was reported to function as a negative regulator of neurotrophin secretion in mouse neurons and as a positive regulator of secretion of a yet to be identified growth factor from muscle cells in flies. Here, we have directly compared the biochemical and functional properties of rat and fly SYT4. We report that rat SYT4 inhibited SNARE-catalyzed membrane fusion in both the absence and presence of Ca2+. In marked contrast, fly SYT4 stimulated SNARE-mediated membrane fusion in response to Ca2+. Analysis of chimeric molecules, isolated C2 domains, and point mutants revealed that the C2B domain of the fly protein senses Ca2+ and is sufficient to stimulate fusion. Rat SYT4 was able to stimulate fusion in response to Ca2+ when the conserved Asp-to-Ser Ca2+ ligand substitution in its C2A domain was reversed. In summary, rat SYT4 serves as an inhibitory isoform, whereas fly SYT4 is a bona fide Ca2+ sensor capable of coupling Ca2+ to membrane fusion.