The use of more reliable and relevant human in vitro models as well as Systems Biology-based approaches to investigate the impact of environmental toxicants such as cigarette smoke (CS) is in line with the recent shift of toxicological assessment strategy to find alternatives for animal testing and to tackle systems level questions in an integrative way. We recently published a comparison study which demonstrated that human organotypic bronchial epithelium-like tissues cultures exposed to CS can recapitulate the biology observed in the bronchial epithelium of smokers (Mathis et al. Am. J. Physiol. Lung Cell. Mol. Physiol. 2013). In the present study, data from large scale mRNA, miRNA, protein analysis, and from immunohistological investigations were captured at different post-exposure time points (0.5h, 2h, 4h, 24h, 48h). By using complementary computational approaches for the analysis of the transcriptomics data (i.e., Gene set enrichment analysis and reverse causal reasoning), a comprehensive picture of the cellular response to CS was obtained which was supported by results from miRNA and phosphoproteomics analysis. Cellular pathways perturbed by CS-exposure were mainly related to a cellular stress response (e.g., oxidative stress), to inflammation (e.g., inhibition of NF-κB and IFN-γ-dependent inflammatory responses), and to proliferation/differentiation. Analysis of the cellular response occurring at different post-exposure time highlighted a transient kinetic of the CS effect after shorter exposure, while for a longer exposure, CS impact was more sustained as seen after 48h post-exposure. Results from omics analyses were further substantiated by immunohistological investigations showing dose-dependent regenerative processes in the exposed tissues.