Since electronic-cigarettes (e-cigarettes) are considered less toxic than conventional tobacco smoking, the use of e-cigarettes has increased, and the market for e-cigarette liquids (e-liquids) is continuously increasing. However, many studies showed that e-cigarettes may cause various harmful effects in lung, oral and heart. In this study, we investigated the effects of e-liquids on otitis media (OM) using human middle ear epithelial cells (HMEECs). Menthol-flavored e-liquid induced significant cell death in HMEECs (IC 50 : 1.45 ± 0.14%) and tobacco-flavored e-liquid led to increase in inflammatory cytokine levels and higher mucin production. Flavored e-liquids decreased the mRNA levels of genes encoding epithelial sodium channels (ENaCs) in HMEECs. Apoptosis and autophagy reactions were induced by exposure of HMEECs to menthol-and tobacco-flavored e-liquids. Tobacco-flavored e-liquids caused a greater increase in the levels of autophagosome marker, LC3-II, compared to menthol-flavored e-liquids, which was followed by cell death. These results demonstrate that flavored e-liquids cause cytotoxicity via apoptosis, autophagy, inflammatory response, and mucin production in HMEECs. The flavors present in e-liquids might be a risk factor for the development of otitis media. Otitis media (OM) is an ear infectious disease and its symptoms include pain, inflammation, and flow of fluid out of the middle ear cavity. Particularly, almost 50% of children experience OM under the age of 3 years 1. The middle ear infection usually presents as acute otitis media (AOM) within one week, but can develop into chronic otitis media (COM). COM persists over 2 weeks, and can cause serious inflammation in the inner ear and hearing loss. OM is typically induced by bacterial and viral infection in the middle ear during the dysfunction of the eustachian tube and nasal cavity 2. However, recent studies have investigated the relationship between environmental factors (smoke, diesel particles, urban particles, and Asian sand dust) and OM incidence 3-6. Electronic cigarettes (e-cigarettes) are available on the market as an alternative to conventional tobacco smoking. E-cigarettes were introduced to help reduce smoking habit by providing nicotine selectively from 0 to 36 mg/ mL 7 in various flavors of e-cigarette liquids (e-liquids). Many users consider e-cigarettes to be safer than tobacco cigarettes, which led to increase in marketing of e-cigarettes for nicotine delivery. However, several animal and human cohort studies have been conducted since 2014 related to the harmful effects of e-cigarettes on human health, including increased tumorigenicity in lung and bladder 8 , cardiovascular system 9 , and alterations in the oral and gut microbiota in humans 10. E-cigarettes vaporize flavor and nicotine-containing fluids through a heating coil in an atomizer, and the user inhales the aerosol. Although e-liquids do not contain harmful agents, such as tar, benzene, carbon monoxide, and formaldehyde, their safety concerns have been raised, except for ...