Synaptic proteins are synthesized in the cell body and transported down the axon by microtubule-dependent motors. We previously reported that KIF1Bβ and KIF1A motors are essential for transporting synaptic vesicle precursors; however the mechanisms that regulate transport, as well as cargo recognition and control of cargo loading and unloading remain largely unknown. Here, we show that DENN/MADD (Rab3-GEP) is an essential part of the regulation mechanism through direct interaction with the stalk domain of KIF1Bβ and KIF1A. We also show that DENN/MADD binds preferentially to GTP–Rab3 and acts as a Rab3 effector. These molecular interactions are fundamental as sequential genetic perturbations revealed that KIF1Bβ and KIF1A are essential for the transport of DENN/MADD and Rab3, whereas DENN/MADD is essential for the transport of Rab3. GTP–Rab3 was more effectively transported than GDP–Rab3, suggesting that the nucleotide state of Rab3 regulates axonal transport of Rab3-carrying vesicles through preferential interaction with DENN/MADD.