Objective: To observe the effect of Haemophilus influenzae colonizing lower respiratory tract on airway inflammation in asthmatic mice and study the related signaling pathway. Methods: Thirty-two C57/B6 mice were sensitized and challenged with ovalbumin (OVA) to establish a mice model of chronic asthma (AC group), and then half of them received intratracheal injection of Haemophilus influenza coated in agar beads to built a mice model of chronic asthma with airway Haemophilus influenza colonization (AS group). Besides, 16 C57/B6 mice were treated with intratracheal injection of Haemophilus influenza (NS group) and 16 C57/B6 mice received intratracheal injection of 0.9% NaCl (NC group). Sixty-four TLR4^-/- mice were treated and grouped as C57/B6 mice. Mice including C57/B6 and TLR4^-/- mice were sacrificed at 7 and 14 days after intratracheal injection, and serum tumor necrosis factor α(TNF-α) content, total number of cells in bronchoalveolar lavage fluid( BALF), and the expression of MyD88 and NF-κB in lung tissue were measured. The indice in AS group were compared with those in the control groups (AC, NS and NC) at corresponding time. Results: The serum level of TNF-α, total number of cells in BALF, and expression of MyD88 and NF-κB in lung tissue were higher in AS group than those in NC and NS group (both in C57/B6 mice and TLR4^-/- mice). Compared with C57/B6 wild-type mice, TLR4^-/- mice in AS group had lower levels of serum TNF-α, decreased numbers of cells in BALF, and down-regulated expression of MyD88 and NF-κB in lung tissue. Conclusions: Lower airway colonization with Haemophilus influenzae may aggravate airway inflammation of asthma by activating the TLR4-MyD88 pathway and promoting expression of NF-κB, which may be one of the important mechanisms of asthma.