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Bisexual individuals may experience pervasive binegativity originating from both heterosexual and lesbian/gay (L/G) individuals as a result of various psychosocial and relational factors. The present study aimed to explore how partner gender is particularly associated with experiences of binegativity from heterosexual and L/G persons and to examine how such experiences are related to internalized binegativity. A total of 350 self-identified cisgender bisexual men and women from across the United States were recruited online for this study. Participants completed an online survey battery assessing levels of both experienced and internalized binegativity. Regression analysis results indicated that binegativity from L/G persons, but not heterosexual persons, was significantly and positively associated with internalized binegativity. A significant interaction between binegativity from L/G persons and partner gender revealed a stronger association among those in same-gender relationships, such that those with same-gender partners who reported binegativity from L/G persons experienced more internalized binegativity than those with other-gender partners. When further examined by gender, these findings appeared to be driven by the relation among women, but not men, as women in same-gender relationships who reported binegativity from L/G persons reported the highest levels of internalized binegativity. Among men, binegativity from heterosexual, but not L/G, persons was significantly related to internalized binegativity independent of partner gender. The present study highlights key gender differences in interpersonal factors related to binegativity and have important implications for clinical practice with bisexual clients facing stigma and advocacy work addressing bisexual discrimination.