An object is to reduce occurrence of defective bonding between a base substrate and a semiconductor substrate even when a silicon nitride film or the like is used as a bonding layer. Another object is to provide a method for manufacturing an SOI substrate by which an increase in the number of steps can be suppressed. A semiconductor substrate and a base substrate are prepared; an oxide film is formed over the semiconductor substrate; the semiconductor substrate is irradiated with accelerated ions through the oxide film to form a separation layer at a predetermined depth from a surface of the semiconductor substrate; a nitrogen-containing layer is formed over the oxide film after the ion irradiation; the semiconductor substrate and the base substrate are disposed opposite to each other to bond a surface of the nitrogen-containing layer and a surface of the base substrate to each other; and the semiconductor substrate is heated to cause separation along the separation layer, thereby forming a single crystal semiconductor layer over the base substrate with the oxide film and the nitrogen-containing layer interposed therebetween.