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Silk fibroin (SF), a natural polymer material possessing excellent biocompatibility and biodegradability, and has been widely used in biomedical applications. In order to explore the behavior of vascular cells by co-culturing on regenerated SF matrix for use as artificial blood vessels, human aorta vascular smooth muscle cells (HAVSMCs) were co-cultured with human arterial fibroblasts (HAFs) or human umbilical vein endothelial cells (HUVECs) on SF films and SF tubular scaffolds (SFTSs). Analysis of cell morphology and deoxyribonucleic acid (DNA) content showed that HUVECs, HAVSMCs and HAFs adhered and spread well, and exhibited high proliferative activity whether cultured alone or in co-culture. Immunofluorescence and scanning electron microscopy (SEM) analysis showed that HUVECs and HAFs co-existed well with HAVSMCs on SF films or SFTSs. Cytokine expression determined by reverse transcription-polymerase chain reaction (RT-PCR) indicated that the expression levels of α-smooth muscle actin (α-SMA) and smooth muscle myosin heavy chain (SM-MHC) in HAVSMCs were inhibited on SF films or SFTSs, but expression could be obviously promoted by co-culture with HUVECs or HAFs, especially that of SM-MHC. On SF films, the expression of vascular endothelial growth factor (VEGF) and platelet endothelial cell adhesion molecule-1 (CD31) in HUVECs was promoted, and the expression levels of both increased obviously when co-cultured with HAVSMCs, with the expression levels of VEGF increasing with increasing incubation time. The expression levels of VEGF and CD31 in cells co-cultured on SFTSs improved significantly from day 3 compared with the mono-culture group. These results were beneficial to the mechanism analysis on vascular cell colonization and vascular tissue repair after in vivo transplantation of SFTSs. [ABSTRACT FROM AUTHOR]