The mucolytic drug ambroxol hydrochloride reduces the production of pro-inflammatory cytokines and the frequency of exacerbation in patients with chronic obstructive pulmonary disease (COPD). However, the inhibitory effects of ambroxol on rhinovirus infection, the major cause of COPD exacerbations, have not been studied. We examined the effects of ambroxol on type 14 rhinovirus (RV14) infection, a major RV group, in primary cultures of human tracheal epithelial cells. RV14 infection increased virus titers and cytokine content in the supernatants and RV14 RNA in the cells. Ambroxol (100 nM) reduced RV14 titers and cytokine concentrations of interleukin (IL)-1β, IL-6 and IL-8 in the supernatants and RV14 RNA in the cells after RV14 infection, in addition to reducing susceptibility to RV14 infection. Ambroxol also reduced the expression of intercellular adhesion molecule-1 (ICAM-1), the receptor for RV14, and the number of acidic endosomes from which RV14 RNA enters the cytoplasm. In addition, ambroxol reduced the activation of the transcription factor nuclear factor kappa B (NF-κB) in the nucleus. These results suggest that ambroxol inhibits RV14 infection partly by reducing ICAM-1 and acidic endosomes via the inhibition of NF-κB activation. Ambroxol may modulate airway inflammation by reducing the production of cytokines in rhinovirus infection.