Adenovirus vectors are attractive delivery systems for vaccines and cancer treatment. Scalable and cost-efficient production technologies are needed to enable manufacturing of safe and efficacious clinical-grade virus. Anchorage-dependent cells cultured in roller bottles or cell factories are commonly used in these processes. However, scale-up using these techniques is complicated and limited by the surface available for cell growth. One alternative is to scale up the production on microcarriers. Another solution is to use suspension-adapted cells, which can facilitate scale-up possibilities. In this work we demonstrate an efficient process for adenovirus production in a single-use stirred-tank bioreactor, using HEK293 cells adapted for suspension culture. Human adenovirus 5 expressing green fluorescent protein (GFP) was used as a model system. By evaluating different cell culture media (CCM) and optimizing the virus propagation in small scale, the process could be successfully established in Xcellerex™ XDR-10 Bioreactor (10 L working volume). This process opens up possibilities for further scale-up to production scale.