A semiconductor device may include a semiconductor substrate including an active region defined by a trench, a device isolation layer provided in the trench to surround the active region, a gate electrode extending in a direction crossing the active region, and formed on the active region and the device isolation layer, and a gate insulating layer between the active region and the gate electrode. The active region may have a first conductivity type, and the device isolation layer may include a first silicon oxide layer on an inner surface of the first trench and a different layer, selected from one of first metal oxide layer and a negatively-charged layer, on the first silicon oxide layer.