Adenovirus infection is a significant cause of ocular, respiratory, and gastrointestinal illness and can spread rapidly. Morbidity is considerable in immune-suppressed individuals and there is significant mortality. There are no effective therapies. During preclinical studies of adenoviral-mediated gene therapy for ocular disorders, we noticed a significant increase in transduction when the target cells were exposed to adenovirus in the presence of ocular vitreous. The vitreous is mainly comprised of water, collagen, and the large polysaccharide hyaluronan. In this paper, we report data that implicate hyaluronan in the adenoviral infectious process and show that interference with the interaction between hyaluronan and its cellular receptor CD44 can block adenovirus transduction in vitro and in vivo.