BACKGROUND: Human rhinovirus (hRV) infections commonly cause acute upper respiratory infections and asthma exacerbations. Environmental cigarette smoke exposure is associated with a significant increase in the risk for these infections in children. OBJECTIVE: To determine the impact of short-term exposure to cigarette smoke on innate immune responses of airway epithelial cells infected with hRV. METHODS: A human bronchial epithelial cell line (HBEC-3KT) was exposed to cigarette smoke extract (CSE) for 30 min and subsequently infected with hRV serotype 1B. Viral-induced cytokine release was measured with AlphaLISA and viral replication quantified by shed viral titer and intracellular viral copy number 24h post-infection. RESULTS: CSE induced a concentration-dependent decrease in CXCL10 (p<0.001) and IFN-beta (p<0.001), with a 79% reduction at the highest dose with an associated 3-fold increase in shed virus. These effects were maintained when infection was delayed up to 24h post CSE exposure. Exogenous IFN-beta treatment at t=0 after infection blunts the effects of CSE on viral replication (p<0.05). CONCLUSION: A single exposure of 30 min to cigarette smoke has a lasting impact on epithelial innate defence providing a plausible mechanism for the increase in respiratory infections seen in children exposed to second-hand tobacco smoke.