Manufacturing cost of an SOI substrate is reduced. Yield of an SOI substrate is improved. A method for manufacturing an SOI substrate includes the steps of irradiating a single crystal semiconductor substrate with ions to form an embrittled region in the single crystal semiconductor substrate, bonding the single crystal semiconductor substrate to a base substrate with an insulating film therebetween, and separating the single crystal semiconductor substrate and the base substrate at the embrittled region to form a semiconductor layer over the base substrate with the insulating film therebetween. In the step of forming the embrittled region, ion species which are not mass-separated are used as the ions and a temperature of the single crystal semiconductor substrate is set to 250° C. or higher at the time of irradiation with the ions.