Objective To investigate effects of 3D co-culture of human keratinocytes (HKC) and human fibroblasts (HFB) under pressure on cell proliferation and collagen synthesis. Methods The HKC and HFB were planted on chitosan-gelatin scaffolds, respectively, for 2 d. The HKC-chitosan-gelatin complex (3D HKC) was cultured at air-liquid interface for 1 d to induce differentiation, and then co-cultured with the HFB-chitosan-gelatin complex (3D HFB) for 12 h. 3.4 kPa pressure was applied on the co-culture group for 24 h. The group of single culture with pressure, the group of single culture without pressure and the group of co-culture without pressure were used as control. HE staining was used to observe distribution and growth of HKC and HFB on chitosan-gelatin scaffolds. MTT method was used to test proliferation of HKC and HFB. Hydroxyproline kit was used to observe collagen concentration of the supernatant fluids. Results HE staining showed that HKC and HFB could grow confluently on chitosan-gelatin scaffolds；3.4 kPa pressure or co-culture both could promote the HKC proliferation and collagen synthesis, while restrain the HFB proliferation and collagen synthesis. Conclusions Pressure and co-culture play an important role in HKC and HFB proliferation and collagen synthesis. This research finding provides some reference for exploring the therapeutic mechanism of hyperplastic scar from clinical operation of resecting scar by transplanting tissue-engineered skin to the wound and then combined with pressure treatment.