Furin is a proprotein convertase that cycles between the plasma membrane, endosomes and the trans-Golgi network (TGN), maintaining a predominant distribution in the latter. Mint3, a member of the Mint protein family, is involved in the signaling and trafficking of membrane proteins. Until now, little has been known about the roles of Mint3 in the localization or trafficking of Furin. Here, using co-immunoprecipitation and immunofluorescence assays, we show that Mint3 interacts with Furin in the Golgi compartment of HeLa cells. Knockdown of endogenous Mint3 expression by RNA interference disrupts the TGN-specific localization of Furin and increases its distribution in endosomes. We further demonstrate that the phosphotyrosine-binding (PTB) domain of Mint3 is essential for the binding of Furin and that this binding affects the TGN-specific localization of Furin. Moreover, mutation studies of Furin indicate that Mint3 regulates Furin distribution mainly through interaction with the acidic peptide signal of Furin. Collectively, these data suggest that the interaction between the PTB domain of Mint3 and the acidic peptide signal of Furin regulates the specific localization of Furin in the TGN.