Previous studies have suggested that the airways of cystic fibrosis (CF) patients have elevated sodium chloride (NaCl) levels due to the malfunctioning of the CF transmembrane conductance regulator protein. For bacteria to survive in this high-salt environment, they must adjust by altering the regulation of gene expression. Among the different bacteria inhabiting the airways of CF patients is the opportunistic pathogen Burkholderia cenocepacia. Previous studies have indicated that B. cenocepacia produces a toxin and cable pili under high osmolar conditions. We used transposon mutagenesis to identify NaCl-regulated genes in the clinical strain B. cenocepacia K56-2. Six transconjugants were induced with increasing NaCl concentration. The DNA flanking the transposon was sequenced and five distinct open reading frames were identified encoding the following putative proteins: an integrase, an NAD-dependent deacetylase, TolB, an oxidoreductase, and a novel hypothetical protein. The collective results of this study provide important information about the physiology of B. cenocepacia when faced with osmotic stress and suggest the identity of significant virulence mechanisms in this opportunistic pathogen.