SNARE-mediated synaptic exocytosis is orchestrated by facilitatory and inhibitory mechanisms. Genetic ablations of Complexins, a family of SNARE-complex-binding proteins, in mice and Drosophila cause apparently opposite effects on neurotransmitter release, leading to contradictory hypotheses of Complexin function. Reconstitution experiments with different fusion assays and Complexins also yield conflicting results. We therefore performed cross-species rescue experiments to compare the functions of murine and Drosophila Complexins in both mouse and fly synapses. We found that murine and Drosophila Complexins employ conserved mechanisms to regulate exocytosis despite their strikingly different overall effects on neurotransmitter release. Both Complexins contain distinct domains that facilitate or inhibit synaptic vesicle fusion, and the strength of each facilitatory or inhibitory function differs significantly between murine and Drosophila Complexins. Our results show that a relative shift in the balance of facilitatory and inhibitory functions results in differential regulation of neurotransmitter release by murine and Drosophila Complexins in vivo, reconciling previous incompatible findings.