Burkholderia pseudomallei is the causative agent of melioidosis, a serious infection endemic in tropical areas, particularly in South-East Asia and northern Australia. Treatment of melioidosis is prolonged due to intrinsic drug resistance as well as the ability of B. pseudomallei to survive intracellular and rapidly spread cell-to-cell. B. pseudomallei uses a variety of mechanisms to subvert the host and survive intracellularly such as inhibition of autophagy, interferon response and abhorrent cytokine production. Previous studies have shown that the folding protein, PpiB, is essential for virulence in a BALB/c mouse model of infection and is cleared from animal organs. Deletion of ppiB results in pleiotropic effects of a number of bacterial virulence determinants including motility, antibiotic susceptibility and multi-nucleated giant cell (MNGC) formation through its Type 6 Secretion System Cluster 1 (T6SS-1). This study aims to determine the key host and bacterial factors which are responsible for this phenotype. Proteomic analysis of infected murine macrophage monolayers was conducted to determine the host response to infection with the wild-type strain (BpsWT) as well as identify key intracellular pathways which were disrupted following deletion of ppiB (BpsΔppiB). This work identified a total of 1506 and 551 host protein changes when infected with BpsWT or BpsΔppiB respectively, compared to BpsWT-infected monolayers. Despite comparable intracellular bacterial numbers, MNGC formation was significantly reduced in cell monolayers infected with BpsΔppiB, corroborated proteomically with a significant decrease of all Type 6 Secretion System Cluster 1 (T6SS-1) proteins. In addition, proteins associated with infection recognition including the interferon response, TNF-α and guanylate binding proteins (Gbp) were increased when infected with BpsΔppiB, proteins which are generally repressed by B. pseudomallei wild-type. This study has shown that the cyclophilin, PpiB, is required for the full pathogenesis of B. pseudomallei through its interactions with multiple virulence determinant such as the flagella and T6SS-1.