An oxide semiconductor layer with excellent crystallinity is formed to enable manufacture of transistors with excellent electrical characteristics for practical application of a large display device, a high-performance semiconductor device, etc. By first heat treatment, a first oxide semiconductor layer is crystallized. A second oxide semiconductor layer is formed over the first oxide semiconductor layer. By second heat treatment, an oxide semiconductor layer including a crystal region having the c-axis oriented substantially perpendicular to a surface is efficiently formed and oxygen vacancies are efficiently filled. An oxide insulating layer is formed over and in contact with the oxide semiconductor layer. By third heat treatment, oxygen is supplied again to the oxide semiconductor layer. A nitride insulating layer containing hydrogen is formed over the oxide insulating layer. By fourth heat treatment, hydrogen is supplied at least to an interface between the second oxide semiconductor layer and the oxide insulating layer.