Burkholderia pseudomallei, a Gram-negative pathogen, is the causative agent of melioidosis in humans. This bacterium can be isolated from the soil, stagnant and salt-water bodies, and human and animal clinical specimens. While extensive studies have contributed to our understanding ofB. pseudomalleipathogenesis, little is known about how a harmless soil bacterium adapts when it shifts to a human host and exhibits its virulence. The bacterium’s large genome encodes an array of factors that support the pathogen’s ability to survive under stressful conditions, including the host’s internal milieu. In this study, we performed comparative transcriptome analysis ofB. pseudomalleicultured in human plasma versus soil extract media to provide insights intoB. pseudomalleigene expression that governs bacterial adaptation and infectivity in the host. A total of 455 genes were differentially regulated; genes upregulated inB. pseudomalleigrown in human plasma are involved in energy metabolism and cellular processes, whilst the downregulated genes mostly include fatty acid and phospholipid metabolism, amino acid biosynthesis and regulatory function proteins. Further analysis identified a significant upregulation of biofilm-related genes in plasma, which was validated using the biofilm-forming assay and scanning electron microscopy. In addition, genes encoding known virulence factors such as capsular polysaccharide and flagella were also overexpressed, suggesting an overall enhancement ofB. pseudomalleivirulence potential when present in human plasma. Thisex vivogene expression profile provides comprehensive information onB. pseudomallei’s adaptation when shifted from the environment to the host. The induction of biofilm formation under host conditions may explain the difficulty in treating septic melioidosis.