RpoS subunit of RNA polymerase is a bacterial alternative sigma factor and major regulator important for response to a variety of stress conditions. However, RpoS-dependent genes in Burkholderia pseudomallei remained undefined. We identified the RpoS regulon of B. pseudomallei using a proteomics approach, which revealed 70 differentially expressed proteins between rpoS(+) and rpoS(-) strains. The RpoS-dependent genes were then classified into 14 functional categories, most of which were related to stress response. We then used Hidden Markov Model (HMM) for prediction of RpoS-dependent promoters in 51 genes encoding 63 down-regulated proteins in rpoS(-) strain and successfully defined such promoters, which were classified into three main groups based upon their consensus sequences. Groups 1 and 2, which had the highest potential, were mostly "stress response" genes, whereas Group 3, with the lowest potential, belonged to genes encoding "hypothetical proteins". The promoter prediction was confirmed by 5'-RACE-PCR sequencing. We also expanded and defined RpoS-controlled genes from RpoS regulon, based on proteomic data, RpoS-dependent promoter prediction, gene organization and operon prediction. We finally confirmed the co-expression of these RpoS-dependent genes in operons using RT-PCR. Our data lead to better understanding of RpoS regulation in B. pseudomallei, whose RpoS regulon differs from other Gram-negative bacteria.