Aim To analyze the effect of Toll like receptor 3 (TLR3) agonist PolyI∶C on the proliferation and apoptosis of human umbilical cord blood-derived endothelial progenitor cells (EPC) and its mechanism. Methods Endothelial progenitor cells were treated with different concentrations of PolyI∶C sequentially, and then the phase of cell cycle and cell apoptosis were tested by flow cytometry. CCK-8 assay was used to detect the effect of TNF-α and IL-1β on the apoptosis of endothelial progenitor cells, and the effect of Caspase 8 inhibitior, Caspase 9 inhibitior and IL-1 receptor 1 neutralizing antibody (anti-IL-1R1) on PolyI∶C-induced apoptosis. Results Compared with the control group, PolyI∶C at high concentrations of 1 and 10 g/L significantly decreased the proportion of cells in S phase and G2/M phase.Moreover, PolyI∶C down-regulated the gene expression of cyclin A, B1, D1 and E, inducing cell cycle arrest in G0/G1 phase. Additionally, PolyI∶C induced the apoptosis of endothelial progenitor cells in a dose-dependent manner. Caspase 8 and Caspase 9 inhibitors did not reduce PolyI∶C-induced apoptosis of endothelial progenitor cells. TNF-α had no effect on apoptosis of endothelial progenitor cells. IL-1β induced cell apoptosis in a dose-dependent manner. Moreover, when endothelial progenitor cells pre-treated with anti-IL-1R1, then re-stimulated with PolyI∶C, the cell apoptosis induced by PolyI∶C was decreased. Conclusions PolyI∶C at high concentrations inhibited endothelial progenitor cells proliferation by inducing cell cycle arrest in G0/G1 phase and inducing cell apoptosis of endothelial progenitor cells. PolyI∶C induced the apoptosis of endothelial progenitor cells through up-regulating the expression of IL-1β via activating TLR3. Endogenous and exogenous apoptosis pathway and TNF-α did not contribute to PolyI∶C-induced cell apoptosis.