We aim to assess sedentary behavior (SB) and its determinants, as well as potential strategies to reduce SB among employees in a tertiary hospital in Singapore, using a mixed-methods approach grounded in the socioecological framework. All employees with email and security guards of a tertiary hospital in Singapore were invited to complete a questionnaire that captured sociodemographics and self-reported domain-specific SB. Environmental influences of occupational SB were assessed using an adapted version of the Checklist for Health Promotion Environments at the Worksite (CHEW). Focus group discussion (FGD) addressed perceptions, barriers, and strategies toward reducing workplace SB. Analyses were performed separately and integrated using an inductive comparative approach. The median occupational sitting time (n = 938) was 300 minutes/day, and highest among administrative staff (administrative, 421 minutes/day; allied health, 300 minutes/day; ancillary, 300 minutes/day; nursing, 120 minutes/day; medical/dental/others, 240 minutes/day; p value: <0.001). The CHEW assessment identified poor physical and information environment contributing to occupational SB. FGDs confirmed an unsupportive environment and elicited barriers such as low SB awareness, nature of work, and workplace norms. Besides environmental approaches, participants suggested having face-to-face communication and social modelling to promote more breaks from sitting. This mixed-methods study among diverse professional groups of a tertiary hospital indicated a large amount of occupational SB, particularly among administrative staff. Raising awareness of the health risks of SB and building a supportive organizational culture, information, and physical environment emerged as significant factors. To reduce occupational SB, multicomponent interventions addressing these diverse factors are warranted.