The aim of this study is to develop human bronchial and nasal epithelium culture models that are relevant to investigate the impact of cigarette smoke (CS) observed in vivo in respiratory tract tissues in contact with inhaled CS. We used two organotypic cultures generated from primary cells derived from non-smoking donors that contain fibroblasts and epithelial cells in order to reproduce as closely as possible the in vivo situation. To mimic the smoking behavior of a moderate smoker during one day, human tissue cultures (bronchial and nasal epithelium) were exposed repeatedly and directly at the air/liquid interface (using the VITROCELL® system) to two doses (high ~ 4.2 µg TPM/cm2 and low ~ 2.2 µg TPM/cm2 per cigarette) of the whole smoke generated by one cigarette or to humidified air (sham). CS exposure was repeated four times with one hour intervals between each cigarette. Various endpoints (e.g., gene and microrna expression, CYP activity, pro-inflammatory markers release, differential cell counts, cytotoxicity measurement) were then captured to assess the baseline (time 0) and early responses of the tissues after exposure (4 hours) as well as the recovery phase (24 and 48 hours). Computational methods such as gene set enrichment analysis were applied to identify the biological perturbations induced by CS exposure in both tissue cultures. A comparison with in vivo datasets from nasal and bronchial epithelial cells obtained from smokers and non-smokers was also undertaken to investigate how close the effect obtained in vitro can reflect the in vivo situation. At the highest CS dose, the global gene expression changes in both tissue cultures are more intense early after exposure and decrease during the recovery phase. Gene set enrichment analysis performed on datasets from both nasal and bronchial tissue cultures captured early after exposure (0 and 4 hours), indicates an induction of genes related to xenobiotic metabolism and inflammation as well as a decreased expression of genes involved in pathways related to fatty acids breakdown and protein synthesis/degradation. In vivo/in vitro comparison of CS effect on gene expression changes related to xenobiotic metabolism shows a good correspondence between nasal in vivo dataset and CS-exposed nasal tissue culture 24 hours after CS exposure. A similar result is observed for CS-exposed bronchial tissue cultures (4 hours) compared to four different datasets derived from bronchial epithelial cells obtained by brushings from smokers and non-smokers. Essential genes involved in the xenobiotic metabolism (e.g. CYP1A1 and CYP1B1) were equally found to be transcriptionally activated in both CS-exposed nasal and bronchial tissue cultures (0 and 4 hours). We describe for the first time the impact of whole CS exposure on a human nasal organotypic in vitro model. By using computational approaches and by capturing systems biology endpoints, various biological perturbations triggered by repeated exposure to CS were observed in both nasal and bronchial in vitro models. Finally, in vitro/in vivo comparison shows the relevance of these two in vitro models in toxicological assessment of CS and could potentially be considered as a good alternative for animal models.