IMPORTANCE: The culture of academic medicine may foster mistreatment that disproportionately affects individuals who have been marginalized within a given society (minoritized groups) and compromises workforce vitality. Existing research has been limited by a lack of comprehensive, validated measures, low response rates, and narrow samples as well as comparisons limited to the binary gender categories of male or female assigned at birth (cisgender). OBJECTIVE: To evaluate academic medical culture, faculty mental health, and their relationship. DESIGN, SETTING, AND PARTICIPANTS: A total of 830 faculty members in the US received National Institutes of Health career development awards from 2006-2009, remained in academia, and responded to a 2021 survey that had a response rate of 64%. Experiences were compared by gender, race and ethnicity (using the categories of Asian, underrepresented in medicine defined as race and ethnicity other than Asian or non-Hispanic White, and White), and lesbian, gay, bisexual, transgender, queer (LGBTQ+) status. Multivariable models were used to explore associations between experiences of culture (climate, sexual harassment, and cyber incivility) with mental health. EXPOSURES: Minoritized identity based on gender, race and ethnicity, and LGBTQ+ status. MAIN OUTCOMES AND MEASURES: Three aspects of culture were measured as the primary outcomes: organizational climate, sexual harassment, and cyber incivility using previously developed instruments. The 5-item Mental Health Inventory (scored from 0 to 100 points with higher values indicating better mental health) was used to evaluate the secondary outcome of mental health. RESULTS: Of the 830 faculty members, there were 422 men, 385 women, 2 in nonbinary gender category, and 21 who did not identify gender; there were 169 Asian respondents, 66 respondents underrepresented in medicine, 572 White respondents, and 23 respondents who did not report their race and ethnicity; and there were 774 respondents who identified as cisgender and heterosexual, 31 as having LGBTQ+ status, and 25 who did not identify status. Women rated general climate (5-point scale) more negatively than men (mean, 3.68 95% CI, 3.59-3.77 vs 3.96 95% CI, 3.88-4.04, respectively, P < .001). Diversity climate ratings differed significantly by gender (mean, 3.72 95% CI, 3.64-3.80 for women vs 4.16 95% CI, 4.09-4.23 for men, P < .001) and by race and ethnicity (mean, 4.0 95% CI, 3.88-4.12 for Asian respondents, 3.71 95% CI, 3.50-3.92 for respondents underrepresented in medicine, and 3.96 95% CI, 3.90-4.02 for White respondents, P = .04). Women were more likely than men to report experiencing gender harassment (sexist remarks and crude behaviors) (71.9% 95% CI, 67.1%-76.4% vs 44.9% 95% CI, 40.1%-49.8%, respectively, P < .001). Respondents with LGBTQ+ status were more likely to report experiencing sexual harassment than cisgender and heterosexual respondents when using social media professionally (13.3% 95% CI, 1.7%-40.5% vs 2.5% 95% CI, 1.2%-4.6%, respectively, P = .01). Each of the 3 aspects of culture and gender were significantly associated with the secondary outcome of mental health in the multivariable analysis. CONCLUSIONS AND RELEVANCE: High rates of sexual harassment, cyber incivility, and negative organizational climate exist in academic medicine, disproportionately affecting minoritized groups and affecting mental health. Ongoing efforts to transform culture are necessary.