E-cigarette usage continues to rise, yet the safety of e-cigarette aerosols is questioned. Using murine models of acute and chronic e-cigarette aerosol inhalation, murine colon transcriptomics and murine and human gut-derived organoids in co-culture models, we assessed the effects of e-cigarette use on the gut barrier. Histologic and transcriptome analyses revealed that chronic, but not acute, nicotine-free e-cigarette use increased inflammation and reduced expression of tight junction markers (TJ). Exposure of murine and human enteroid-derived monolayers (EDMs) to nicotine-free e-cigarette aerosols alone or in co-culture with bacteria, also cause barrier-disruption, downregulation of TJ protein, enhanced inflammation in response to infection. These data highlight the harmful effect of “non-nicotine” component of e-cigarette on gut barrier. Considering the importance of an intact gut barrier for host fitness, and the impact of gut mucosal inflammation on a multitude of chronic diseases, these findings are broadly relevant to both medicine and public health.