Alcohol use disorder (AUD) is a common mental disorder that may cause dementia and mood disorders such as anxiety. The dentate gyrus (DG) is a key brain region for dementia as well as anxiety, while the underlying mechanisms of DG-based behavioral impairments induced by ethanol are not fully understood. Here we report the DG-based behavior inhibition using the ethanol vapor exposure procedure combined with behavioral tasks. The results indicate that single ethanol vapor exposure leads to working memory impairments and decreased anxiety-like behaviors 24 h after single ethanol vapor exposure. Notably, the c-Fos staining shows that the hippocampal DG is overactivated by ethanol administration, which implies DG plays a pivotal role in mediating the working memory and anxiety-like behaviors induced by ethanol vapor exposure. This links overactivation effects of ethanol vapor on hippocampal DG functions to ethanol-induced behavior inhibition, suggesting that future studies concerning ethanol-induced cognitive and mood disorders may focus more on the abnormal activity of this brain region.