Background: Although circadian disruption plays an essential role in chronic airway diseases, the upstream mechanisms remain largely unexplored. Microbiota regulates gut molecular clock, yet its relationship with lung clock is unknown. Aims: To investigate the effects of airway microbiota on lung clock gene expression in a mouse model. Methods: Mice were intranasally exposed to cigarette smoke for 3 months. Microbiota depletion was done by antibiotic treatment and microbiota transplantation by intratracheal transfer of bacterial pellets isolated from bronchioalveolar lavage fluids of donor mice to antibiotic-treated mice. All animals were sacrificed at Zeitgeber time (ZT0). Lung tissue was used for 16S rRNA gene sequencing and RNA sequencing. Results: Cigarette smoke induced an altered airway microbiota confirmed by beta diversity analysis (p=0.033), with enriched Bacteroides and Parabacteroides (both q=0.039). Transcriptomic analysis identified 96 differentially expressed genes and circadian rhythm was the most profoundly enriched KEGG pathway when comparing smoke vs. air-exposed mice. Specifically, 3 clock genes (BMAL1 [q=2.50×10^-9], CLOCK [q=4.00×10^-4] and NFIL3 [q=2.00×10^-4]) were downregulated while DBP (q=9.25×10^-5) upregulated in smoking mice. Notably, depletion of microbiota in smoking mice restored the expression of BMAL1 (q=6.96×10^-19), CLOCK (q=5.87×10^-9) and NFIL3 (q=7.09×10^-4) while downregulated DBP (q=1.98×10^-15). Finally, transplantation of airway microbiota from smoking mice recapitulated BMAL1 downregulation (q=0.001) and DBP upregulation (q=0.034) in recipient mice. Conclusion: Cigarette smoke-induced airway dysbiosis results in abnormal expression of lung clock genes.