The harm of cigarette smoke (CS) exposure to both the lower and upper respiratory tracts is widely known. Electronic cigarette (EC) exposure has been suggested to exert less harm compared with CS exposure. Many studies have assessed the potential toxicity of ECs in vitro. Yet, most studies tested the effects of the liquid formulations applied directly on cell cultures but not the effects of the formulations applied as a vapor/aerosol. In this study, using human organotypic buccal and small airway epithelial cultures, we examined the effects of an acute exposure to whole aerosol generated by a novel EC device, using MESH™ technology, and to whole mainstream CS. Nine independent exposure experiments were conducted. In each experiment, cultures were exposed at the air-liquid interface to undiluted aerosol of “Classic Tobacco” flavor generated from the novel EC for 112 puffs or to diluted CS for the same puff number in Vitrocell® exposure systems. Deposited nicotine and carbonyls in the exposure chamber were measured as exposure markers. Using systems toxicology, we complemented histological analysis with quantitative analysis of molecular changes within a 48-hour recovery period following exposure (global expression profiles of both mRNA and miRNA and targeted protein profiles, including secretory proteins). Tissue damage was not seen in cultures exposed to the EC aerosol despite resulting in greater concentrations of deposited nicotine. Unlike CS exposure, EC aerosol exposure resulted in negligible concentrations of carbonyls in the exposure chamber. Among the secreted proteins analyzed, many more proteins regulating inflammatory response were detected following exposure to CS exposure than to the EC aerosol. The global mRNA profiles pointed toward alterations in mechanisms related to cellular fate, proliferation, stress, and inflammatory response following CS exposure. These alterations were noticeably less and reverted back to those in cultures exposed to air 48 hours following exposure to the EC aerosol. Fewer changes in the miRNA and protein profiles were detected following EC aerosol exposure than CS exposure in both cultures. Overall, the findings suggested that relative to the impacts of CS exposure, much smaller impacts were detected following undiluted EC aerosol exposure in buccal and small airway epithelial cultures.