Colistin is a last-line treatment option for infections caused by multi-drug resistant strains of Acinetobacter baumannii. The known mechanisms of colistin resistance involve changes to the lipopolysaccharide (LPS). These include the addition of phosphoethanolamine (PEtn) and/or galactosamine, or complete inactivation of lipid A biosynthesis. Previously, we characterized a colistin-susceptible A. baumannii parent strain and a colistin-resistant mutant isolated from a patient before and after colistin treatment, respectively, and showed that colistin resistance was conferred by the insertion of ISAba125 within hns, a gene that encodes a HNS-family transcriptional regulator. To understand this mechanism of colistin resistance and to identify the HNS regulon in A. baumannii, we performed transcriptional analyses of both the pre- and post-treatment isolates and compared the RNA expression profiles of the strains. Transcriptomic analyses identified 171 differentially expressed genes (143 genes were up regulated and 28 genes were down regulated in the colistin-resistant strain). The genes controlled either directly or indirectly by HNS including those involved in motility (type I pili), biofilm formation, chemotaxis and the type VI secretion system. Importantly, the PEtn transferase gene, eptA (a homologue of pmrC) showed increased expression (5.2 Fold change) in the hns mutant whilst the expression of the Pmr operon (pmrA, pmrB and pmrC), known to be involved in colistin-resistance via PEtn modification of lipid A, remained unchanged. Expression of eptA on the plasmid pWH1266 in the colistin-susceptible isolate of A. baumannii conferred colistin-resistance compared to a colistin-susceptible isolate harbouring pWH1266 vector alone. These results indicate that the colistin-resistance in the hns mutant is mediated, at least in part, via increased expression of eptA which is predicted to result in additional PEtn modification of lipid A. This is the first time an HNS-family transcriptional regulator has been associated with the regulation of a PEtn transferase and associated colistin resistance.