AIM: Establishing functional circulation on time is crucial to dental pulp tissue regeneration. Mesenchymal stem cells (MSCs) could act as mural cells to stabilize newly formed blood vessels, accelerating anastomosis. Our preliminary study found that direct coculture of dental pulp stem cells (DPSCs) and human umbilical vein endothelial cells (HUVECs) significantly enhanced Activin A secretion. This study aimed to disclose the dynamic patterns of Activin A expression and its regulation on vascular formation and stabilization. METHODOLOGY: DPSCs and HUVECs were cocultured directly at a ratio of 1:1 for 3 and 6 days. Activin A and Follistatin expression were evaluated by qRT-PCR and ELISA. HUVECs were exposed to 100 ng/mL Activin A or the conditioned medium (CM) generated from DPSC monoculture and DPSC-HUVEC coculture, respectively. HUVEC proliferation, migration, tube formation and angiogenic sprouting were assessed. In parallel, membrane-bound vascular endothelial growth factor receptors (mVEGFR1 and mVEGFR2) and soluble VEGFR1 (sVEGFR1) were analysed at days 3 and 6. RESULTS: Activin A expression and secretion were elevated time-dependently during DPSC-HUVEC coculture. Follistatin expression decreased in DPSC-HUVEC coculture while the ratio of Activin A/Follinstain increased significantly. Activin A treatment did not promote DPSC towards smooth muscle cell (SMC)-specific differentiation, while Activin A and DPSC+HUVEC-CM suppressed HUVEC proliferation, migration, tube formation and sprouting. Activin A and DPSC+HUVEC-CM treatment markedly increased mVEGFR1 expression and sVEGFR1 secretion, suppressing HUVEC vascular formation. Activin A IgG partially reversed the effects of DPSC+HUVEC-CM on HUVECs by decreasing VEGFR1 expression and increasing vessel formation. Activin A pretreatment downregulated VEGF-triggered VEGFR2 phosphorylation of HUVECs. INHBA knockdown DPSCs disrupted the stabilization of the preformed HUVEC vascular tube network. CONCLUSION: DPSC-HUVEC direct coculture upregulates Activin A secretion, interrupting VEGF receptors' balance in HUVECs to suppress HUVEC angiogenic sprouting and enhance vascular stabilization. These findings provide novel insights into the paracrine interactions on vascular stabilization of DPSC-HUVEC direct coculture.