Provided is a method of manufacturing a substrate for liquid ejection head, including: forming a groove portion by etching on one surface side of a silicon substrate, the groove portion being formed so as to surround a portion at which a liquid supply port is to be formed on an inner side of the groove portion; forming a protective layer on the one surface side of the silicon substrate, the protective layer being formed inside the groove portion and on an outer side of the groove portion; and forming the liquid supply port by subjecting the silicon substrate to crystal anisotropic etching treatment with use of the protective layer as a mask.