This study investigated the factors that cause job stress among male flight attendants in a female-dominated airline organization, as well as the impact of job stress on their mental health and turnover intention. It also attempted to determine whether perceived family support, perceived organizational support, and job positions had moderating effects on male flight attendants’ job stress. Six job stress factors were identified through focus group interviews and a literature review. A survey was conducted from 1 January to 2 February 2022 to validate the research model, and 188 valid samples were used for statistical analysis. This study discovered that gender differences in communication, relationship conflict with colleagues, hierarchical organizational culture, and role overload had a direct impact on male flight attendants’ job stress. Job stress was found to have a negative impact on mental health and a positive impact on turnover intention. Perceived organizational support was also found to reduce job stress. This study is notably the first to address stress encountered by male flight attendants at work. It offers new directions for future airline personnel management and research. It also presents practical implications, such as the development of training and personnel management programs for male flight attendants.