An object is to provide a single crystal semiconductor layer with extremely favorable characteristics without performing CMP treatment or heat treatment at high temperature. Further, an object is to provide a semiconductor substrate (or an SOI substrate) having the above single crystal semiconductor layer. A first single crystal semiconductor layer is formed by a vapor-phase epitaxial growth method on a surface of a second single crystal semiconductor layer over a substrate; the first single crystal semiconductor layer and a base substrate are bonded to each other with an insulating layer interposed therebetween; and the first single crystal semiconductor layer and the second single crystal semiconductor layer are separated from each other at an interface therebetween so as to provide the first single crystal semiconductor layer over the base substrate with the insulating layer interposed therebetween. Thus, an SOI substrate can be manufactured.