Background: Physical inactivity is an increasing public health issue which has been associated with several chronic illnesses. However, despite strong evidence indicating the health benefits of physical activity, many university staff and students tend to be physically inactive. The aim of this study was to examine the perceived barriers to and enablers of physical activity among staff and students in a university setting, using the Theoretical Domains Framework (TDF). Methods: This was a qualitative study carried out in a university in the East Midlands’ region of the United Kingdom. Eight group interviews were conducted with 6 male and 15 female (mean age= 40.5 ± 10.6 years) university staff with different job roles (e.g., academic, administrative, cleaning and catering staff), and 12 male and 7 female (mean age= 28.6 ± 4.7 years) students at different stages of study (e.g., undergraduate, postgraduate, and international students). Interviews were audio recorded, transcribed verbatim and imported into Nvivo12 software, responses were coded using the TDF where theory-driven deductive content analysis was used for data analysis. Results: The evolving themes from the group interviews mapped to the 14 domains of the theoretical domains framework as enablers and/or barriers to physical activity. However, 71% of these themes mapped to six major domains of the TDF (i.e., environmental context and resources; intentions; social influences; knowledge; beliefs about capabilities; and social/professional role and identity), and thus established as the major enablers of and barriers to physical activity among university staff and students.Conclusions: These findings suggest that the capability, opportunity and motivation of university staff and students to engage in physical activity are influenced by several enablers and barriers. This study therefore provides a theoretical foundation to inform the development of bespoke interventions to increase physical activity among inactive university staff and students, as well as in other settings.