Mucociliary clearance is a critical innate defense system responsible for clearing up invading pathogens including bacteria and virus. Although the right amount of mucus is good, excessive mucus causes airway obstruction and tends to precipitate disease symptoms. Rhinovirus (RV) is a common cold virus that causes asthma and chronic obstructive pulmonary disease exacerbation. Mucus overproduction has been linked to the pathogenesis of RV-induced diseases and disease exacerbations. However, the molecular mechanism is not clear. In this study, using one of the major airway mucin-MUC5AC as marker, we found that both major and minor groups of RV induced mucin production in primary human epithelial cells and cell line. RV1A (a minor group of RV) could induce mucous cell metaplasia in vivo. Viral replication was needed for RV-induced mucin expression, and this induction was also dependent on TLR3, suggesting the involvement of double-stranded (ds) RNA signaling. Indeed, dsRNA alone could also induce mucin expression. TLR3-mediated mucin induction was negatively regulated by MyD88, and only partially dependent on TRIF, which suggests a departure from well-documented TLR3 signaling paradigm that mediates inflammatory and other innate defense gene inductions. In addition, TLR3 signaling activated epidermal growth factor receptor (EGFR) through inductions of the expression of EGFR ligands (transforming growth factor-alpha and amphiregulin), which in turn activated EGFR-ERK signaling and mucin expression through an autocrine/paracrine loop. This novel coupling of antiviral defense machinery (i.e., TLR3) and major epithelial proliferation/repair pathway (i.e., EGFR) might play an important role in viral-induced airway remodeling and airway disease exacerbation.