Bordetella pertussis, the causative agent of whooping cough or pertussis, is an obligate human pathogen with multiple high-affinity iron transport systems. Maximal expression of the dedicated heme utilization functions encoded by the hurIR bhuRSTUV genes requires an iron starvation signal to relieve Fur repression at the hurIR promoter-operator and an inducing signal supplied by heme for HurI-mediated transcriptional activation at the bhuRSTUV promoter. The BhuR outer membrane receptor protein is required for heme uptake and for heme sensing for induction of bhuRSTUV transcription. It was hypothesized that heme utilization contributed to the success of B. pertussis as a pathogen. In this study, virulence attenuation resulting from inactivation of the B. pertussis heme system was assessed using mixed infection competition experiments in a mouse model. As a measure of in vivo fitness, the ability of a B. pertussis heme utilization mutant to colonize and persist was determined relative to that of an isogenic coinfecting wild-type strain. Relative fitness of the mutant strain declined significantly after 7 days postinfection and continued to decline throughout the remainder of the 28-day infection time course. In parallel infections using inocula supplemented with an inducing 2 μM concentration of hemin chloride, hemin coadministration augmented the competitive advantage of the wild-type strain over the mutant. The results confirm that heme utilization contributes to the pathogenesis of B. pertussis in the mouse infection model and indicate that heme utilization may be most important for adaptation to host conditions existing during the later stages of infection.