Objective To research on cell seeding for large-area tissue construction in a bag bioreactor. Method A tissue engineered 3-D model system was developed by using human fibroblasts and porous polyethylene terephthalate (PET) polymer scaffolds. The effects of shaking speed and initial cell concentration in suspension on cell seeding kinetic, cell seeding efficiency, initial seeding density and cell distribution within the scaffolds were studied. Result Under the experimental conditions, the process of human fibroblasts seeding to large-area PET generally followed the first order reaction kinetics. And at low initial cell concentration, the seeding efficiency and initial seeding density decreased with the increasing shaking speed, while at high initial cell concentration the results were reverse. The cell distributing uniformity all decreased with shaking speed increased for all different cell concentrations, but there was no significant difference in uniformity between different initial cell concentrations under the same shaking intenslty. Conclusions The horizontal shake seeding method used in a bag bioreactor for large-area scaffold was approved feasible, which provides basic foundation for further optimizing cell seeding method for such large-area tissue construction.