Burkholderia cenocepacia is a ubiquitous environmental organism associated with life-threatening opportunistic infections in Cystic Fibrosis patients. Within these patients, B. cenocepacia proliferates in host macrophages (1) by establishing a membrane-bound replicative niche known as the B. cenocepacia-containing vacuole (BcV). To enhance our understanding of the host-pathogen interactions governing BcV formation, we have established proteomic approaches enabling the characterisation of intracellular B. cenocepacia, as well as the spatial changes within infected THP-1 macrophages. Utilising centrifugation-based enrichment of intracellular B. cenocepacia, we demonstrate that intracellular derived bacteria possess unique proteome patterns pre (3hr) and post (24hr) BcV formation, supporting that intracellular replication is associated with discrete B. cenocepacia lifestyles. Focusing on proteins exclusively produced during intracellular replication, mutagenesis and infection assays reveal several proteins that contribute to the survival of B. cenocepacia within macrophages, as well as a novel factor essential for replication. To understand host changes associated with the formation of the BcV, we have established a centrifugation-based fractionation spatial proteomics workflow (2) and benchmarked this against whole proteome analysis. We find that cellular fractionation improves proteomic coverage by 22% and accurately discriminates proteins based on their subcellular localisation. Applying this approach to infected THP-1 cells, we observe not only protein level changes but evidence for protein redistribution in response to the formation of the BcV. Overall, this work identifies novel B. cenocepacia factors required for intracellular replication, as well as host changes associated with the formation of the BcV, enhancing our understanding of the biology of B. cenocepacia infections.