Because Varp (VPS9-ankyrin-repeat protein)/Ankrd27 specifically binds two small GTPases, Rab32 and Rab38, which redundantly regulate the trafficking of melanogenic enzymes in mammalian epidermal melanocytes, it has recently been implicated in the regulation of trafficking of a melanogenic enzyme tyrosinase-related protein 1 (Tyrp1) to melanosomes. However, the functional interaction between Rab32/38 and Varp and the involvement of the VPS9 domain (i.e. Rab21-GEF domain) in Tyrp1 trafficking have never been elucidated. In this study, we succeeded in identifying critical residues of Rab32/38 and Varp that are critical for the formation of the Rab32/38·Varp complex by performing Ala-based site-directed mutagenesis, and we discovered that a conserved Val residue in the switch II region of Rab32(Val-92) and Rab38(Val-78) is required for Varp binding activity and that its point mutant, Rab38(V78A), does not support Tyrp1 trafficking in Rab32/38-deficient melanocytes. We also identified two critical residues for Rab32/38 binding in the Varp ANKR1 domain and demonstrated that their point mutants, Varp(Q509A) and Varp(Y550A), do not support peripheral melanosomal distribution of Tyrp1 in Varp-deficient cells. Interestingly, the VPS9 domain point mutants, Varp(D310A) and Varp(Y350A), did support Tyrp1 trafficking in Varp-deficient cells, and knockdown of Rab21 had no effect on Tyrp1 distribution. We also found evidence for the functional interaction between a vesicle SNARE VAMP7/TI-VAMP and Varp in Tyrp1 trafficking. These results collectively indicated that both the Rab32/38 binding activity and VAMP7 binding activity of Varp are essential for trafficking of Tyrp1 in melanocytes but that activation of Rab21 by the VPS9 domain is not necessary for Tyrp1 trafficking.