The cystic fibrosis (CF) lung is notable for the presence of steep oxygen gradients. Burkholderia cenocepacia, a bacterium that causes chronic infections in people with CF, must adapt to this low-oxygen niche to successfully colonise. To investigate whether hypoxia drives adaptation of B. cenocepacia towards chronic infection in CF, an early CF infection isolate (P1E) was continually cultured in either hypoxia (6% oxygen) or normoxia (21% oxygen) for 22-days in-vitro. A proteomic analysis of hypoxia-adapted cultures (HACs) relative to normoxia-adapted cultures (NACs) revealed significant changes in abundance consistent with those observed in a late infection isolate (P1L) relative to P1E. Whole-genome sequencing analysis, including comprehensive SNP profiling, was also conducted.  A phenotypic analysis revealed attachment of HACs to CF epithelial cells was increased 2-fold relative to NACs (p<0.0001), consistent with observations in PIL. Protease activity was also confirmed to be 2.5-fold higher in HACs relative to both NACs and P1E (p<0.0001), consistent with P1L. The HACs also showed 2-fold greater survival (p<0.05) in CF-macrophages relative to NACs, following the trend observed in chronic infection isolates. Stable colony variants were isolated from HACs, including a novel “doughnut” variant which exhibited 3-fold more attachment to CF epithelial cells relative to P1E (p<0.05). Protease activity was also significantly elevated in the novel variants (p<0.0001). Notably, the doughnut variants were more virulent than P1E in the Galleria mellonella acute infection model. These data highlight that hypoxia is driving stable changes in B. cenocepacia, leading to diversification and contributing to adaptation to chronic infection.