In order to avoid detection of residual animal serum and improve recombinant adenovirus productivity in the preparation of recombinant adenovirus production, Trex-293 human embryonic kidney adherent cells adapted to growth in suspension using animal serum-free CD293 medium in spinner flask and observed the growth patterns and virus yields in different conditions. The experimental results show that adherent cultured Trex-293 cell is able to be adapted to suspension culture with serum-free CD293 medium in spinner flask. Initial growth time to a stable state is longer than that of adherent cell culture, but since then they rapidly growth. The cell viability was low at early stage, maintained 90% viability at 14 d after culture. The suspension cells seeded at 4-5?105cells /mL reached 2?106 cells/mL by 3-4 d of culture, and reached 3?106 cells/mL at 21 d after culture, doubling time was about 32 h. The yield of recombinant adenovirus infected in suspension cells with ratio of 200 VP/cell and cell densities of 2 ? 106 cells / mL is higher than that of infection ratio of 500 VP/cell and 1000 VP/cell, and it also higher than that of in adherent cells. These results concluded that adherent Trex-293 can be adapted to growth in suspension conditions with serum-free CD293 medium in spinner flask. For recombinant adenovirus infects in suspension cells, it helps to improve the yield of virus and the yield also higher than adherent cells under the condition of an infection ratio of 200 VP/cell, harvest at 48 h post-infection and cell viability of 50%.