<p>Hypoxia is a hallmark of solid tumors and represents a major barrier for effective cancer therapies, including oncolytic virotherapy. While adenoviruses are widely studied as oncolytic agents, the impact of tumor-associated hypoxia on viral infection and spatial spread remains incompletely understood. Here, we investigated how oxygen availability influences adenovirus infection in two-dimensional (2D) cultures and three-dimensional (3D) tumor spheroids. We confirmed that cell lines commonly used in adenovirus research (HEK293A, A549), as well as KP4 pancreatic cancer cells, exhibited a physiological response to hypoxia. In KP4 monolayers, hypoxia strongly reduced adenoviral protein production. To model oxygen gradients found in solid tumors, we established stable KP4 spheroids and performed spatial analysis of HAdV5_GFP infection. When virus was added during spheroid formation and hypoxia development, infection was largely restricted to the well-oxygenated outer rim. In contrast, inoculation of virus under normoxia prior to spheroid formation resulted in a more uniform distribution of infected cells throughout the spheroid. Together, our findings demonstrate that hypoxia not only suppresses adenoviral replication in cell culture but also shapes the spatial pattern of infection in 3D tumor models, highlighting the importance of hypoxia-relevant 3D systems in preclinical evaluation of oncolytic adenoviruses.</p>