The Type VI Secretion System (T6SS) is an important virulence system that exists in many bacterial pathogens, and has emerged as a potent mediator of pathogenicity in Acinetobacter baumannii. In this study, we inactivated one of the T6SS components vgrG (valine-glycine repeat G) gene in A. baumannii ATCC 19606 and constructed a complementation strain. BEAS-2b human alveolar epithelial cells was adopted to assess bacterial adhesion, and wild female BALB/c mice were used for in vivo experiments to assess the bacterial killing ability to host. Upon deletion of the vgrG gene, increased antimicrobial resistance to ampicillin/sulbactam, but reduced resistance to chloramphenicol were observed. The vgrG mutant strain showed lower growth rate, reduced eukaryotic cell adherence and impaired lethality in mice. However, the vgrG mutant strain is not implicated in biofilm formation. Our study suggests that the Type VI Secretion System core component VgrG contributes to both virulence and antimicrobial resistance in A. baumannii ATCC 19606.