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To date, few studies have addressed attitudes toward transgender individuals. In addition, little is known about health care providers’ (HCP) attitudes toward sexual minorities. The aim of the present study is to compare attitudes toward homosexual and transgender individuals between gender dysphoric individuals (GDs), general population controls (C) and HCP. A total of 310 subjects were considered, including 122 GDs (63 transwomen and 59 transmen), 53 heterosexual HCP (26 males and 27 females) and 135 C. Participants completed the Modern Homophobia Scale (MHS) and the Attitudes Toward Transgendered Individuals Scale (ATTI) in order to assess attitudes toward gay men and lesbian women and toward transgender individuals, respectively. In addition, GDs completed the Gender Identity/Gender Dysphoria Questionnaire (GIDYQ-AA) and ATTI to measure, respectively, gender dysphoria levels and internalized transphobia. Religious attitudes were evaluated by means of the Religious Fundamentalism Scale (RFS), and Discrimination and Stigma Scale (DISC-12) was used to measure perceived discrimination. (1) Men showed significantly higher levels of homophobia and transphobia when compared to women (p< 0.001); (2) perceived discrimination was higher in lesbian women compared to gay men and in transwomen compared to transmen (p< 0.001 and p< 0.05, respectively); and (3) religious fundamentalism was associated with both homophobia and transphobia (both p< 0.001). Our results underline the need to promote awareness and acceptance of the sexual minorities, who are more at risk of discriminatory attitudes, which are strongly dependent on religious precepts and dogma.