Rab GTPases are key regulators of intracellular membrane traffic acting through their effector molecules. Rabaptin-5 is a Rab5 effector in early endosome fusion and connects Rab5- and Rab4-positive membrane compartments owing to its ability to interact with Rab4 GTPase. Recent studies showed that Rabaptin-5 transcript is subjected to extensive alternative splicing, thus resulting in expression of Rabaptin-5 isoforms mostly bearing short deletions in the polypeptide chain. As interactions of a Rab GTPase with different effectors lead to different responses, functional characterization of Rabaptin-5 isoforms becomes an attractive issue. Indeed, it was shown that Rab GTPase effector properties of Rabaptin-5 and its α and δ isoforms are different. This work focused on another Rabaptin-5 isoform, Rabaptin-5γ. Despite its ability to interact with Rab5, endogenously produced Rabaptin-5γ was absent from early endosomes. Rather, it was found to be tightly associated with trans-Golgi network and partially localized to a Rab4-positive membrane compartment. The revealed intracellular localization of Rabaptin-5γ indicates that it is not involved in Rab5-driven events; rather, it functions in other membrane transport steps. Our study signifies the role of alternative splicing in determination of functional activities of Rab effector molecules.