As bisexual individuals in the United States (U.S.) face significant health disparities, researchers have posited that these differences may be fueled, at least in part, by negative attitudes, prejudice, stigma, and discrimination toward bisexual individuals from heterosexual and gay/lesbian individuals. Previous studies of individual and social attitudes toward bisexual men and women have been conducted almost exclusively with convenience samples, with limited generalizability to the broader U.S. population. Our study provides an assessment of attitudes toward bisexual men and women among a nationally representative probability sample of heterosexual, gay, lesbian, and other-identified adults in the U.S. Data were collected from the 2015 National Survey of Sexual Health and Behavior (NSSHB), via an online questionnaire with a probability sample of adults (18 years and over) from throughout the U.S. We included two modified 5-item versions of the Bisexualities: Indiana Attitudes Scale (BIAS), validated sub-scales that were developed to measure attitudes toward bisexual men and women. Data were analyzed using descriptive statistics, gamma regression, and paired t-tests. Gender, sexual identity, age, race/ethnicity, income, and educational attainment were all significantly associated with participants' attitudes toward bisexual individuals. In terms of responses to individual scale items, participants were most likely to “neither agree nor disagree” with all attitudinal statements. Across sexual identities, self-identified other participants reported the most positive attitudes, while heterosexual male participants reported the least positive attitudes. As in previous research on convenience samples, we found a wide range of demographic characteristics were related with attitudes toward bisexual individuals in our nationally-representative study of heterosexual, gay/lesbian, and other-identified adults in the U.S. In particular, gender emerged as a significant characteristic; female participants’ attitudes were more positive than male participants’ attitudes, and all participants’ attitudes were generally more positive toward bisexual women than bisexual men. While recent population data suggest a marked shift in more positive attitudes toward gay men and lesbian women in the general population of the U.S., the largest proportions of participants in our study reported a relative lack of agreement or disagreement with all affective-evaluative statements in the BIAS scales. Findings document the relative lack of positive attitudes toward bisexual individuals among the general population of adults in the U.S. and highlight the need for developing intervention approaches to promote more positive attitudes toward bisexual individuals, targeted toward not only heterosexual but also gay/lesbian individuals and communities.