Background: The low physical activity is a challenge in adults. A majority of adults spend many hours each week at work, so the workplace is a suitable site for health promotion and implementation of physical activity programs. This qualitative study was implemented to identify worksite physical activity barriers and facilitators from the perspective of the employees of the Iran University of Medical Science. Methods: In the present qualitative content analysis, five focus group discussions were held with the participation of 68 personnel of Iran University of Medical Sciences. Purposive sampling with maximum diversity was carried out for the study. Results: The analysis of the data led to the identification of three general themes, including challenges and barriers, strategies, and incentives (facilitators). The four main categories of challenges and barriers included policy-making and legislation, organizational factors, structural factors, and personal factors. Most barriers identified by the participants were placed in the personal factors and organizational factors categories. The strategies for increasing physical activity were identified in the following three categories: Policy-making and legislation, organizational factors, and environmental factors. The majority of the strategies proposed were placed in the organizational factors and policy-making and legislation categories.Conclusions: Increasing physical activity in the workplace as a strategy for the general promotion of physical activity in people requires interventions in different areas, especially with regard to organizational factors and policy-making and legislation.