Multidrug resistant (MDR) bacterial infections are increasing in frequency worldwide, which coupled with a lack of new drugs, places enormous pressure on treatment regimes. Therefore, understanding how bacterial pathogens circumvent treatment is urgently required. Acinetobacter baumannii is a Gram-negative opportunistic hospital-acquired pathogen of which many strains are MDR. As A. baumannii uses a multifactorial process to cause disease these elements are likely to be under stringent genetic control. To provide insight into these regulatory networks we examined the role of a response regulatory element (RR) within the MDR clinical A. baumannii 04117201 isolate. The RR protein is part of a two-component system (TCS), consisting of a membrane-bound histidine kinase and a cognate cytosolic RR, working in concert to respond to environmental changes. The RR stkR of the TCS stkRS was deleted using site-specific recombination. The resulting ∆stkR strain was assessed for alterations in bacterial call adherence, surface hydrophobicity, and its resistance profile to selected antibiotics including colistin, known as a “last resort” drug. Compared to the parent, the ∆stkR strain demonstrated a 2-fold increase in eukaryotic cell adherence, increased cell surface hydrophobicity and a 2-fold increase in colistin resistance. Protein analysis of the ∆stkR strain grown in the presence of increasing concentrations of colistin identified several proteins differentially expressed compared to the parent strain. Previous studies have shown colistin resistance to be due to major alterations in the lipid A of resistant A. baumannii strains; examination of the surface polysaccharides of the ∆stkR strain revealed only minor changes in this region. Analysis of high throughput RNA sequencing revealed a number of genes that are differentially expressed and has potentially identified the source of the increased colistin resistance, representing a novel mechanism in A. baumannii.