Human 293S cells culture for recombinant adenovirus production is traditionally carried out in batch at a maximum of 6 × 105 cells/mL. A previous report demonstrated that fed-batch, applied to the adenovirus/293S cells system, improves the volumetric production of viral proteins by increasing the cell density at which cells can be infected, up to 2 × 106 cells/mL, without reducing the per-cell yield of product. To increase this cell density limit, the adenovirus production was performed in a perfusion system where the cells were separated by means of a tangential flow filtration device. 293S cell growth to 14 × 106 cells/mL was achieved in 10 days, at a medium renewal rate of 1 volume of medium per reactor volume and day (VVD). For adenovirus production, three 293S cell cultures were perfused at 1 VVD in parallel and infected at an average density of 8 × 106 cells/mL. One of the cultures was set at 37 °C and the two others at 35 °C. After a rapid initial cell loss, the average cell density stabilized at 5.75 × 106 cells/mL, 12 h postinfection, which was 8 times higher than the cell density in the batch control. This allowed the production of 3.2 × 109 infectious viral particles/mL (IVP/mL) at 37 °C and 7.8 × 109 IVP/mL at 35 °C, this last result being 5.5 times higher than the control. To our knowledge, this nonconcentrated titer is the highest value that has ever been published for adenovirus vector production. These observations lead to the conclusion that perfusion is an efficient tool to maintain, at high cell density, a specific production rate level sufficient to increase significantly the adenovirus volumetric production. Furthermore, it shows that perfusion at 35 °C can improve viral titer by 2.4-fold compared to 37 °C, in accordance with a previous study on adenovirus batch production.