A method of manufacturing semiconductor device includes preparing a substrate having a first surface and a second surface opposite to the first surface. A first insulation layer is formed on the second surface. A sacrificial layer is formed on the first insulation layer. An opening is formed to penetrate through the substrate and extend from the first surface to a portion of the sacrificial layer. A second insulation layer is formed on an inner wall of the opening. A plug is formed to fill the opening. The sacrificial layer is removed to expose a lower portion of the plug through the second surface.