Cigarette smoking is one of the major risk factors for the development of lung cancer. However, little is known about the effects of nicotine, a major constituent of cigarette smoke, on lung epithelial cells in the context of lung tumorigenesis. In order to investigate whether nicotine elicits differential effects on mechanisms promoting or leading to carcinogenesis, human immortalized non-tumorigenic BEAS-2B and tumorigenic BZR bronchial epithelial cells were continuously exposed to low, medium, and high concentrations of nicotine (10, 100, and 1000 nM) for four weeks. Proliferation, apoptosis, and expression of metalloproteases were assessed weekly by real-time impedance measurements using the xCELLigence® platform, high-content imaging, and Luminex technology. Gene expression analysis was performed on nicotine-treated and untreated cells and collected weekly using microarrays together with a computational network approach. In addition, soft agar assays were performed at the end of the treatment period of examine anchorage independence. Nicotine did not increase the proliferation of immortalized BEAS-2B or BZR cells as determined by cell counting. Real-time impedance data indicate a small but transient pro-proliferative nicotine effect on BEAS-2B cells at week 3. Nicotine treatment had no impact on staurosporine-induced apoptosis of BEAS-2B and BZR cells. Furthermore, nicotine treatment did not increase the levels and activity of metalloproteases. Systems toxicological analysis indicated a small but non-consistent impact when treating BEAS-2B cells with nicotine, while BZR cells seemed to be more responsive to nicotine than BEAS-2Bcells. Finally, anchorage-independent growth was not observed in BEAS-2B or BZR cells when treated for four weeks with nicotine. In conclusion, chronic nicotine treatment of immortalized non-tumorigenic BEAS-2B and tumorigenic BZR cells did not promote cell proliferation, suppress apoptosis, or initiate any mechanisms that favor tumorigenesis. Longer exposures might be necessary to elucidate the contribution of nicotine to cancer promotion and progression.