Ethyl carbamate (EC) is thought to be a toxicant that widely exists in cigarette smoke and polluted air, as well as fermented food and alcoholic beverages. However, the mechanism and approach to treat hepatic damage after EC exposure remain unclear. Here, we first found that EC caused decreased cell viability, reactive oxygen species (ROS) overproduction and glutathione (GSH) depletion in normal human hepatocytes L02 cells. Excessive ROS generation was found to be one of the major reasons for cell cytotoxicity of EC treatment. Furthermore, increased ROS levels also promoted autophagy, a lysosomal degradation process, which was confirmed by detection of LC3-II expression and puncta in GFP-RFP-LC3 transfection assay. Autophagy inhibitor chloroquine (CQ) pretreatment led to decreased cell viability and higher ROS levels compared with EC group, suggesting that autophagy protected EC-treated cells against oxidative stress and cytotoxicity. Notably, we observed increased lysosomal biogenesis and activation of transcription factor EB (TFEB), a master regulator of lysosomal generation, in the process of autophagy. Taken together, we unveiled a novel mechanism of hepatotoxicity and endogenous potent protection of TFEB-mediated autophagy against decreased cell viability and redox disturbance under EC exposure in normal human hepatocytes.