The interaction of bacteria with the human respiratory epithelial mucosa is a crucial factor in the pathogenesis and in persistence of infections with non-typeable H. influenzae (NTHi). Analysis of NTHi metabolic endproducts during growth on a tissue culture-related medium has demonstrated that the bacteria consume ribose present in nucleosides such as inosine, making ribose a major nutrient. However, whether the ability to grow on pentose sugars affects virulence has not been studied yet. In this investigation, the physiological effects of mutations in rbsB, that encodes the substrate binding protein of the NTHi ribose ABC transporter, and rbsK and rpiA, that encode two enzymes involved in the pentose phosphate pathway, ribokinase (RbsK) and ribose-5-phosphate isomerase (RpiA) were investigated. Mutant strains were constructed, and complemented using both single and multi-gene complementation. Phenotypical validation showed that rbsB and rbsK mutants were unable to grow on ribose, while the rpiA mutant showed an ~2-fold reduction in growth compared to the wildtype. The mutations also reduced growth on ribose from uridine, indicating that ribose, and not uracil is used as a carbon source. Phenotypic microarrays confirmed a reduced ability to grow on ribose and other pentoses for the rbsK mutant. The inability to efficiently use ribose as a carbon source created other phenotypic alterations such as a ~10% reduction (p<0.0001) in ATP content, but had no effect on hypochlorite resistance. Infection assays using 16HBE14 bronchial epithelial cells, unexpectedly, showed no significant changes relative to the WT. In contrast, in mouse bone marrow macrophage infections, the rbsB and rbsK mutants showed 2- and 4- fold reductions, respectively, in intracellular survival at 2h post infection. Overall, the findings indicate that ribose utilization supports NTHi virulence, and appears to be particularly important for oxidative stress survival, i.e. in contact with phagocytic immune cells.