The Gram-negative bacterium Acinetobacter baumannii is an increasingly prominent nosocomial pathogen with multi-drug resistance. Effective antibiotic treatment is urgently required to treat this rapidly evolving, globally distributed bacterium. The Type VI secretion system (T6SS) of A. baumannii is a complex nanomachine that delivers protein effectors into the environment or directly into host cells and/or competing bacteria. Investigation of post-translational modifications, namely protein phosphorylation, could be used to identify new therapeutic targets to effectively control this bacterium. By modification of protein function, phosphorylation is known to play an important role in bacterial virulence, adaptation and resistance. In initial proteomic studies, the serine/threonine/tyrosine phospho-secretome was investigated in two A. baumannii strains during both planktonic and biofilm growth: reference strain ATCC17978 and the virulent multi-drug resistant AB0057 strain. Five phosphosites were identified during biofilm growth for ATCC17978 in the haemolysin coregulated protein (Hcp); a sheath protein and core component of the T6SS, known to be essential for T6SS-mediated interbacterial competition. To examine the role of these phosphosites in the function of Hcp, site-directed mutagenesis of the selected phosphosites was performed in the virulent and highly similar A. baumannii strain AB307-0294. Alanine, a non-phosphorylated amino acid, was used to individually replace the phosphorylated amino acids. The modified Hcp proteins were expressed in an A. baumannii AB307-0294 hcp mutant, and T6SS function assessed via Hcp secretion. It was shown that one of these phosphosites, Ser18, was essential for formation of an active type VI secretion system during planktonic growth. However, when Ser18 was substituted with a phosphomimetic (aspartic acid), Hcp secretion was not restored. A number of the other identified A. baumannii Hcp phosphosites are currently under investigation to assess their role in Hcp secretion and T6SS function.