Burkholderia pseudomallei is the causative agent of melioidosis, a disease endemic in South-East Asia and northern Australia. Mortality rates in these areas are high even with antimicrobial treatment, due to few options for effective therapy. Therefore there is a requirement to identify anti-bacterial targets for the development of novel, effective treatments. Cyclophilins are a family of highly conserved enzymes shown to be important in multiple cellular processes. Cyclophilins catalyse the cis-trans isomerization of xaa-proline bonds, a rate limiting step in protein folding which has been shown to play a role in bacterial virulence. B. pseudomallei strain K96243 encodes for a cytoplasmic cyclophilin B gene, ppiB, the role of which was investigated in this study. A cyclophilin B-null mutant strain, BpsΔppiB, was generated and characterised. In vitro macrophage invasion studies showed that BpsΔppiB had 36.8-fold reduction in intracellular numbers at 9 hours, which upon further investigation using immunofluorescence showed that there was a decrease in cell-to-cell spread and fusion of macrophages into Multi-nucleated Giant Cells (MNGC). BpsΔppiB is avirulent in the BALB/c mouse infection model with all survivors clearing infection. BpsΔppiB also displayed a reduction in motility and biofilm formation as well as an increase in susceptibility to oxidative stress and antibiotics. To determine the mechanisms behind the reduction in virulence, global proteomic analysis was conducted and demonstrated the loss of PpiB leads to widespread disruption of the proteome with 734 proteins undergoing statistically significant alterations. The loss of PpiB leads to decreased abundance of multiple virulence determinants including flagellar machinery, chemotaxis detection systems, capsular polysaccharide and alterations in Type-VI secretion systems-associated proteins. The alteration in these protein levels supports our hypothesis that PpiB is important in maintaining proteome homeostasis and thus virulence in B. pseudomallei.