Electronic nicotine delivery systems (ENDS) dispense nicotine by heating a solution (e-liquid) and generating an aerosol that is then inhaled by the user. ENDS have been previously reported by some as less harmful than conventional cigarettes. However, available data regarding the toxicity of ENDS devices and e-liquids are limited and further studies are needed to fully understand their biological effects. In this study, we investigated the toxicity of nonflavored e-liquids in primary human bronchial epithelial cells using a systems toxicology approach. The toxicity of e-liquids was compared to that of nicotine alone. Cells were exposed to solutions containing different proportions of propylene glycol (PG), vegetable glycerin (VG), and nicotine. The results were compared with those of exposing cells to nicotine alone. Multiparametric indicators of cellular toxicity were measured via real-time cellular analysis and high-content screening. This study was complemented by a whole transcriptome analysis, followed by computational approaches to identify and quantify perturbed molecular pathways. Exposure to PG and VG resulted in inhibited cell proliferation and cytotoxicity. These effects were proportional to the PG content in the mixture and consistent with the presence of hyperosmotic shock in exposed cells. Addition of nicotine increased the toxicity of the PG/VG mixtures and activated biological pathways ultimately leading to apoptosis/necrosis. Similar responses were observed on exposure to nicotine alone. Taken together, the results indicate that the toxicity of nicotine-containing PG/VG mixtures is similar to that of nicotine alone.