From the perspective of social identity theory, some social psychologists have suggested that heterosexual men espouse negative attitudes toward gay men as a defensive mechanism against threats to their gender self-esteem. The purpose of the present study is to examine whether this gender self-esteem defense theory of sexual prejudice applies among heterosexual men and women in Japan. Our results in principle supported the gender self-esteem defense theory for heterosexual men. For heterosexual women, however, the results tended to be contradictory to the theory. The more positive heterosexual women’s gender self-esteem was, the less negative was their attitude toward lesbians. But this link tended to disappear when they were informed that no biological differences exist between heterosexuals and homosexuals. Our findings suggest that heterosexual men and women maintain their gender self-esteem in different manners: Heterosexual men maintain positive gender self-esteem by embracing negative attitudes toward gay men, but heterosexual women do not. Heterosexual women’s gender self-esteem may be related to expressing tolerance for sexual minorities.