A national seroprevalence study was performed to determine the prevalence of Haemophilus influenzae type b (Hib) antibodies in England and Wales in 2009, when Hib disease incidence was the lowest ever recorded. A total of 2,693 anonymised residual sera from routine diagnostic testing submitted by participating National Health Service hospital laboratories were tested for Hib anti-polyribosyl-ribitol phosphate (PRP) IgG antibodies using a fluorescent bead assay. Median anti-PRP IgG concentrations were highest in toddlers aged 1–4 years (2.65 µg/ml), followed by children aged 5–9 years (1.95 µg/ml). Antibody concentrations were significantly lower after this age, but were still significantly higher among 10–19 year-olds (0.54 µg/ml) compared with adults aged >20 years (0.16 µg/ml; p<0.0001). Half of the adults (51%) did not have Hib antibody concentrations ≥0.15 µg/ml, the level considered to confer short-term protection. Thus, the current excellent Hib control appears to be the result of high anti-PRP antibody concentrations in children aged up to 10 years, achieved through the various childhood vaccination campaigns offering booster immunisation. The lack of seroprotection in adults emphasises the importance of maintaining control of the disease and, most probably carriage, in children, therefore raising the question as to whether long-term routine boosting of either pre-school children or adolescents may be required.