Acinetobacter baumannii is an opportunistic pathogen that is emerging as one of the leading causes of hospital acquired infections. Despite the medical importance of A. baumannii, the mechanisms by which this bacterium mediates disease are poorly understood. Type VI secretion systems (T6SS) are macromolecular machines required by many Gram-negative bacteria for virulence and/or interbacterial competition. Currently, it is known that A. baumannii encodes a functional T6SS as determined by the secretion of the major T6SS structural component Hcp and that in certain strains this system is required for interbacterial competition. We used single crossover insertional mutagenesis to inactivate the gene encoding TssM, a core T6SS structural component, in the A. baumannii strain AB307-0294 and showed that Hcp secretion was highly reduced in the mutant strain. Interbacterial competition assays using an E. coli prey strain revealed AB307-0294 requires a functional T6SS to effectively kill competing E. coli. Furthermore, in vitro invasion assays revealed the mutant strain to be significantly attenuated in its ability to invade A549 lung epithelial cells. These results provide the first evidence that, in addition to providing an interbacterial advantage, the T6SS of A. baumannii may also play a role in pathogenesis by mediating the invasion of pneumocytes.