Acinetobacter baumannii is a nosocomial pathogen that causes a range of infections, the most clinically important of which are ventilator-associated pneumonia and bloodstream infections. Antibiotic resistance in this species is a global health concern, with many emerging pan-drug resistant clinical isolates. The persistence and survival of A. baumannii in nosocomial settings is further aided by the presence of a type VI secretion system (T6SS). This bacterial nanomachine allows for the delivery of toxic effector proteins directly into target cells. Delivered effector proteins target essential bacterial structures, and therefore allow for the elimination of competitors. The system consists of a membrane complex, a base plate, and a contractile needle. This needle, composed of repeating Hcp hexamers, has a tip composed of a trimer of VgrG proteins that is typically capped with a PAAR protein. The clinical isolate A. baumannii AB307_0294 delivers three such toxic effectors: Tse15, Tde16 and Tae17. These are delivered via non-covalent interactions with the cognate T6SS tip proteins VgrG15, VgrG16 and VgrG17, respectively. In this study, we showed that VgrG17 is a master regulator, crucial for the assembly and activity of the A. baumannii AB307_0294 T6SS and use bacterial two hybrid analysis to show that the C-terminal ~50 amino acids of VgrG17 interact with the N-terminal 162 amino acids of Tae17. Alanine scanning mutagenesis of the C-terminal region of VgrG17 identified two amino acids crucial for Tae17 delivery; the first time such specific interactions have been observed for effector delivery. Furthermore, construction of a Tae17::MBP fusion protein allowed us to confirm that the N-terminal 162 amino acids of Tae17 were sufficient for VgrG17-dependent delivery of novel engineered effectors. Such detailed understanding of the interactions required for the delivery of toxic effectors by the T6SS will allow in the future to engineer novel therapeutic effectors and deliver them directly into cells of interest.