Objective: The current study was aimed to mimic the physiology of human pulmonary vascular using an improved contact co-culture method containing human pulmonary artery endothelial cells(HPAECs) and human pulmonary artery smooth muscle cells (HPASMCs). Methods: HPAECs and HPASMCs were seeded on the opposite sides of a porous Transwell membrane that allowed formation of cell-cell contacts. The adhesion of HPAECs was determined by inverted phase contrast microscope and Hoechst staining. Flow cytometry was used to analyze the apoptosis of HPAECs and HPASMCs in different media and serum concentrations. The markers of PAECs and PASMCs were detected separately by immunofluorescence for identifying the characterization of the two kinds of cells. Results: The data from Hoechst staining showed that gelatin-precoated Transwell membrane facilitated the adhesion of HPAECs. Moreover, the apoptosis of HPAECs was increased under the hyperosmotic pressure. Compared with DMEM and RMPI 1640 medium, the endothelial cell medium(ECM) was more suitable for the contact co-culture system. Additionally, ECM with 1% endothelial cell growth supplement(ECGs) and 2% fetal bovine serum(FBS) promoted the growth of both HPAECs and HPASMCs. Moreover, co-culture adjustment showed no significant impact on characterization of PAECs and PASMCs in the aspect of cell morphologies and specific markers. Conclusion: ECM maintaining 1% ECGs and 2% FBS is better for setting HPAECS-HPASMCs contact co-culture system to mimic the physiology of human pulmonary vascular and to investigate the interactions between the two kinds of cells.