The Gram-negative saprophytic bacterium Burkholderia pseudomallei, the aetiologic agent of melioidosis, is not a human commensal, and its presence in clinical specimens generally represents disease requiring antibiotic treatment. Melioidosis ranges in severity, and typically presents in either an acute or chronic form. A 27-year ongoing study of melioidosis cases in the Northern Territory has documented just two highly unusual cases of “chronic-carriage” melioidosis, whereby patients did not display obvious melioidosis symptoms but harboured B. pseudomallei well beyond all other cases. The longest case ever documented, P314, continues to be infected with B. pseudomallei since first being diagnosed with melioidosis in 2000. We traced the within-host evolution of B. pseudomallei retrieved over multiple time points from P314 using both whole-genome sequencing (WGS) and transcriptomics (RNA-seq). WGS revealed dramatic genetic loss after just two years of infection, with a ~4% reduction in genome size that has been stably maintained over time; ~50% of these loci have also been lost in the equine-adapted clone, B. mallei. Approximately 60% of the 144 single-nucleotide polymorphism (SNP) and insertion-deletion sites are non-synonymous, demonstrating a strong signal of selection. Multiple lineages were observed, some of which have persisted over time despite multiple courses of antibiotics. Nonsynonymous mutations were seen in lipopolysaccharide and capsule loci, antibiotic resistance genes, sigma factors and virulence loci, showing that these loci are targeted during long-term survival in the mammalian host. RNA-seq comparison of the initial isolate vs. an isolate retrieved just 15 months later revealed dramatic changes in differential expression, with 1,209 genes significantly up- or down-regulated, despite only 11 SNP and insertion-deletion mutations separating these isolates. Latter isolates demonstrate significantly slower growth and are morphologically distinct from the earlier strains, consistent with major genomic and transcriptomic changes. This unparalleled melioidosis case has answered many previously unknown questions about B. pseudomallei, and provides a fascinating resource for understanding co-adaptation of bacterial pathogens to their hosts.