It is an object to manufacture a semiconductor device in which a transistor including an oxide semiconductor has normally-off characteristics, small fluctuation in electric characteristics, and high reliability. First, first heat treatment is performed on a substrate, a base insulating layer is formed over the substrate, an oxide semiconductor layer is formed over the base insulating layer, and the step of performing the first heat treatment to the step of forming the oxide semiconductor layer are performed without exposure to the air. Next, after the oxide semiconductor layer is formed, second heat treatment is performed. An insulating layer from which oxygen is released by heating is used as the base insulating layer.