ABSTRACT: Importance:With a renewed focus on medical professionalism, an opportunity exists to better define its standards and application to meet the needs of an increasingly diverse workforce given the important association between interprofessional behavior and patient care. Objective:To examine the context of how professionalism is operationalized and perceived in diverse health care work and learning environments. Design, Setting, and Participants:A qualitative mixed-methods analysis of survey data collected from February to April 2015, was conducted followed by analysis of narrative data collected in June 2017. The setting was 2 health systems and 4 health professional and graduate schools. Participants were faculty, trainees, staff, and students (3506 survey respondents and 52 narratives) affiliated with the University of Pennsylvania and the University of Pennsylvania Health System. Data analysis was conducted in 2018 and 2019. Exposures:Independent variables included the following respondent characteristics: gender identity, sexual orientation, race/ethnicity, position, generational age group, length of employment at institution, disability status, belief system or religion, and primary site of work or study. Main Outcomes and Measures:Survey questions were used to assess participants' perception and experiences of professionalism in the workplace as measured by a 5-point Likert-type scale. Results:For the survey, there were 3506 respondents from a pool of 18 550 potential respondents (18.9% response rate). Of 3506 survey respondents, 2082 of 3231 (64.4%) were women, 331 of 3164 (10.5%) identified as gender or sexual minority groups, and 360 of 3178 (11.3%) were non-Hispanic Black individuals. In adjusted analyses, women compared with men (adjusted odds ratio [aOR], 1.8; 95% CI, 1.4-2.3) and Asian individuals (aOR, 2.0; 95% CI, 1.7-2.3) and Hispanic individuals (aOR, 2.0; 95% CI, 1.4-2.7) compared with non-Hispanic White individuals were more likely to value institutional professionalism. In addition, gender identity and sexual minority groups compared with heterosexual respondents (aOR, 1.5; 95% CI, 1.2-1.8) and non-Hispanic Black individuals compared with non-Hispanic White individuals (aOR, 1.3; 95% CI, 1.2-1.4) were statistically significantly more likely to consider changing jobs because of unprofessional behavior at work. The qualitative analysis of narratives revealed that marginalized populations (including but not limited to women, gender and sexual minority groups, racial/ethnic minority groups, those who identify as having a disability, and religious minority groups) reported (1) greater infringements on their professional boundaries, as well as increased scrutiny over their professional actions, and (2) a tension between inclusion vs assimilation. Conclusions and Relevance:The findings of this study highlight the need for health care organizations to revisit how they define and operationalize professionalism to improve inclusivity.