Background: Exercise training at work has the potential to improve employees’ productivity, health, and well-being. However, exercise interventions for healthcare workers in hospitals may be challenged by time pressure and the ongoing workflow with patient care. Objective: The aim was to identify barriers and facilitators for participation in exercise training during work in a hospital department. Methods: Eight semi-structured interviews of 13 individuals were conducted with hospital employees from different staff groups who participated in 12 weeks of exercise twice weekly. The data analysis was a thematic approach based on the Theoretical Domains Framework and the COM-B factors in the Behavior Change Wheel. Results: Barriers and facilitators varied between different groups. Barriers included limited structure, busyness, and a discouraging culture. Facilitators included gaining a feeling of community and psychological and physical well-being. Seven contextual subthemes were vital for successful implementation of exercise in a hospital setting: sharing of knowledge and information; involvement; administration and structure; culture; individualization; purpose and objective; and incentives. Conclusions: The informants appreciated exercise training during work. Inpatient departments’ informants found it difficult to participate in the intervention, whilst those with more administrative tasks found it easier. This study identified barriers and facilitators vital for a successful implementation of an exercise training intervention in a hospital department. The study explains how future interventions can improve reach, adoption, and implementation of exercise training interventions to hospital staffs.