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Abstract Background Low physical activity in adulthood is a major public health challenge. The majority of adults spend many hours each week at work, and workplace thus becomes a suitable location in which to promote health and implement physical activity programs. This qualitative study was conducted to identify the barriers and facilitators of worksite physical activity from the perspective of the employees of Iran University of Medical Sciences. Methods In this qualitative thematic analysis, five focus group discussions were held with the participation of 68 staff members of Iran University of Medical Sciences who had been selected by purposive sampling with maximum diversity. 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.