Human rhinovirus (HRV) triggers exacerbations of asthma and chronic obstructive pulmonary disease. Cigarette smoking is the primary risk factor for the development of chronic obstructive pulmonary disease, and 25% of individuals with asthma smoke. Smokers experience both longer and more severe colds. We previously showed that cigarette smoke extract (CSE) inhibited HRV-induced expression of a range of epithelial antiviral molecules. Here, we use CXCL10 as a model antiviral gene to examine the mechanisms by which CSE inhibits epithelial antiviral immunity. HRV-induced CXCL10 transcription depends on activation of NF-kB and IFN-regulatory factor-1 (IRF-1), and we now also implicate two signal transducer and activator of transcription (STAT) consensus sequences in the CXCL10 promoter in HRV-induced CXCL10 expression. CSE inhibited HRV-induced activation and nuclear translocation/binding of both NF-kB, and IRF-1 to their respective recognition sequences in the CXCL10 promoter. HRV also induced formation of complexes at the STAT region in the CXCL10 promoter, and HRV-induced activation of STAT-1 was inhibited by CSE. In addition, CSE inhibited HRV-induced chromatin accessibility around the transcriptional start site of the CXCL10 promoter. Although CSE inhibited HRV-induced expression of both the viral double-stranded RNA sensors, retinoic acid-inducible gene-I and melanoma differentiation-associated gene (MDA) 5, only specific short interfering RNA (siRNA) to MDA5, but not nontargeting siRNA, or siRNA to retinoic acid-inducible gene-I, inhibited HRV-induced CXCL10 induction. We conclude that CSE reduces chromatin accessibility and inhibits viral signaling via NF-kB, IRF-1, STAT-1, and MDA5. Thus, we show that CSE can simultaneously modulate multiple pathways linked to innate immune responses to HRV infection.