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Though often conceptualized as sharing a common cultural ingroup, trans individuals face barriers to acceptance and social equality that lesbian, gay, and bisexual individuals do not. Traditionally gender-segregated settings (e.g., bathrooms) are contexts in which trans people seem more likely to experience intolerance and discrimination. This study tested the hypothesis that attitudes toward trans people are more negative in gender-segregated than gender-integrated settings, and it explored possible individual differences that could explain this discrepancy in attitudes. Heterosexual cisgender participants recruited from Amazon’s Mechanical Turk (n = 189) completed an online survey that included measures of attitudes toward trans people in gender-segregated and gender-integrated settings, transphobia, ambivalent sexism, and beliefs that the world is a dangerous place. Analyses indicated that attitudes do differ by context, with participants reporting significantly more negative attitudes in gender-segregated settings. Additional analyses suggest that this difference could be in part explained by belief in a dangerous world and benevolent sexist attitudes. These findings suggest that the unsubstantiated safety concerns espoused by some opponents of trans rights may be sincere; however, they may also be more reflective of a subjective and fearful worldview than of objective reality.