Smoker’s oral mucosa is subject to cigarette smoke (CS)-induced cytological, genomic, and transcriptional changes that could potentially lead to the development of mouth disease. To characterize cs effects, we used two human organotypic in vitro models of the buccal and gingival epithelia (MatTek®). Both in vitro models were exposed at the air-liquid interface to fresh, diluted CS (one reference cigarette 3R4F at a time, with Health Canada smoking regimen and nicotine doses of 0.279 mg/l or 0.557 mg/l) on 4 occasions with 1h intervals between exposures where the tissues were kept in the incubator under appropriate culture conditions. Systems biology endpoints (gene and miRNA expression) were determined at time 0 following the 4th exposure and 4h, 24h, 48h after exposure to investigate time- and dose-dependent CS effect on both tissues. Other endpoints (e.g. cytotoxicity, pro-inflammatory marker release, cytochrome P450 (CYP) activity, histology) were also measured for some time points. The release of pro-inflammatory markers such as vegf and mmp-1 was up-regulated in both cs-exposed buccal and gingival tissues 24h after exposure. However, only gingival tissues showed a significant up-regulation of CYP1A1/CYP1B1 activity 48h after exposure. By using computational approaches and by capturing systems biology endpoints, various perturbations of biological processes (e.g. inflammation, proliferation, cellular stress,) triggered by repeated CS exposure were observed in both buccal and gingival in vitro models. We describe here for the first time the impact of CS exposure on human buccal and gingival organotypic models using various approaches combining systems biology, biological network models, computational methods and standard endpoints.