Metabolism of xenobiotics into reactive intermediates often contributes to toxicity, thus the impact of exposure on the host xenobiotic metabolism machinery is often utilized for toxicity assessment. For the assessment of inhaled agents, organotypic airway epithelial models become relevant as they are cultured at the air-liquid interface and resembling better the airway tissue in vivo. Different from conventional cigarette smoking, by which over 6000 compounds are generated from combustion, heating tobacco instead of burning produces lower concentrations of harmful and potentially harmful constituents. In this study, we compare the impact of smoke from 3R4F reference cigarettes to that of the aerosol from a tobacco heating system (THS)2.2, a heat not burn technology-based candidate modified risk tobacco product (MRTP), on the xenobiotic metabolism response. Three human epithelial organotypic tissue cultures (nasal, oral and bronchial) were exposed to cigarette smoke (CS) or THS2.2 aerosol for 28 min. Subsequently, CYP1A1/1B1 activity and gene expression profiles were generated at various post-exposure time points. The results indicate that, at the comparable nicotine concentrations, the activity of CYP1A1/1B1 and gene expression of various CYPs were higher after CS exposure than after exposure to THS2.2 aerosol in all three cultures. Using a xenobiotic metabolism network model and systems biology approach, the transcriptomic data illustrate a greater impact on the xenobiotic metabolism in CS-exposed tissues than THS2.2-exposed tissues. Collectively, our observation in the three in vitro cultures suggests that the xenobiotic metabolism responses can discern the potential toxicity profiles associated with CS from that associated with THS2.2 aerosol.