The purpose of this qualitative descriptive study is to describe how women academic department chairs in emergency medicine, surgery, and anesthesiology experience humor in the workplace. Interviews were conducted with 35 women department chairs in academic medicine from 27 institutions that aimed to describe women's leadership emergence. The data from the primary study yielded rich and revealing themes involving participants' experiences with humor in the context of their leadership roles, justifying a secondary analysis focusing specifically on these experiences. Relevant remarks were extracted, coded, and summarized. Participants discussed two broad types of humor-related experiences. First, they described how they responded to aggressive gender-based humor directed at themselves or their colleagues by tolerating it or expressing disapproval. This humor includes demeaning quips, insulting monikers, sexist jokes, and derogatory stories. Participants often did not confront this humor directly as they feared being rejected or ostracized by colleagues. Second, they described how they initiated humor to address gender-related workplace issues by highlighting gender inequalities, coping with sexual harassment and assault, and managing gender-based leadership challenges. Participants felt constrained in their own use of humor because of the need to be taken seriously as women leaders. Women leaders in academic medicine use humor to confront gender-related issues and experience aggressive gender-based humor in the workplace. The constraints placed on women leaders discourage them from effectively confronting this aggressive gender-based humor and perpetuating gender inequities. Eliminating aggressive gender-based humor is needed to create safe and equitable work environments in academic medicine.