Nontypeable Haemophilus influenzae is a common cause of respiratory tract disease and initiates infection by colonizing the nasopharynx. The H. influenzae Hap adhesin is an autotransporter protein that was discovered because it promotes intimate interaction with human epithelial cells. Hap contains an extracellular domain called Hap(s) that has adhesive and protease activity and an outer membrane domain called Hap(beta) that serves to present Hap(s) on the surface of the cell. Hap(s) purified from nontypeable H. influenzae strain P860295 was used to immunize BALB/c mice intranasally. Immunization stimulated significant mucosal and serum anti-Hap(s) antibody titers, which were augmented by the addition of mutant cholera toxin (CT-E29H) as an adjuvant. Immunization was associated with a marked reduction in the density of nasopharyngeal colonization when mice were challenged with a heterologous strain of nontypeable H. influenzae. These results suggest that intranasal immunization with Hap formulated with CT-E29H may be a valuable vaccine strategy for the prevention of nontypeable H. influenzae disease.