Acinetobacter baumannii is a Gram-negative nosocomial human pathogen, which has become a serious public health issue largely due to its “resist and persist” phenotype. Multidrug efflux systems encoded by this organism are one of the major contributors to its broad antimicrobial resistance. Multidrug efflux pumps in bacterial pathogens contribute to various cellular activities beyond drug resistance, such as biofilm formation, secondary metabolism, and expression of virulence factors. However, there is a limited knowledge about the role of efflux pumps in the pathogenesis of A. baumannii. AceI is a multidrug transport protein and the prototype of the recently described Proteobacterial Antimicrobial Compound Efflux (PACE) family of efflux pumps. It is conserved in the core genome of A. baumannii and can confer resistance to the biocide chlorhexidine, which is commonly used as an antiseptic in wound dressings, hand washes, and mouthwashes. Recently we have reconstituted the AceI protein in proteoliposomes and demonstrated that it confers efflux of diamines such as putrescine and cadaverine, which may represent the physiological substrates of AceI. In this study, we sought to explore the role of AceI in pathogenesis in A. baumannii. We examined the role of AceI in virulence in the mouse septicemia model and observed an almost complete loss of virulence in an aceI-inactivated mutant compared to the parental strain. Furthermore, the aceI inactivated mutant showed reduced serum survival, reduced biofilm formation, reduced capsule production and an inability to produce long-chain acyl homoserine lactones as quorum sensing signals, suggesting a potential involvement of AceI in the pathophysiology of A. baumannii.