Aim To observe the regulation of GTP cyclohydrolase I (GTPCH I)/tetrahydrobiopterin (BH4) pathway on function of endothelial progenitor cells in hypertensive patients. Methods Nineteen hypertensive patients and nineteen healthy volunteers were observed. Endothelial progenitor cells were isolated from peripheral blood, cultured and identified. The in-vitro migration, proliferation, adhesion activity and in-vivo reendothelialization function of endothelial progenitor cells were detected. The model of arterial injury in nude mice was established and endothelial progenitor cells from hypertensive patients and healthy volunteers were transplanted to evaluate the reendothelialization capacity of endothelial progenitor cells. In addition, the mRNA expression of GTPCH I/BH4, and the expression of nitric oxide (NO), cyclic guanosine monophosphate, and thrombospondin-1 (TSP-1) mRNA levels of endothelial progenitor cells were measured. Furthermore, RNA interfection, gene transfection and drug inhibition of GTPCH I/BH4 were carried out to demonstrate its role in the regulation of endothelial progenitor cell function. Results The in-vitro migration, proliferation, adhesion activity and in-vivo reendothelialization function of endothelial progenitor cells were significantly decreased in hypertensive patients. In parallel, the expression of GTPCH I/BH4, NO and cyclic guanosine monophosphate of endothelial progenitor cells were reduced, while the TSP-1 mRNA level was elevated. When blocking the GTPCH I/BH4 pathway, the in-vitro migration, proliferation, adhesion activity and in-vivo reendothelialization function of endothelial progenitor cells from hypertensive patient and healthy volunteers were both weakened. Conclusion This study revealed that GTPCH I/BH4 pathway regulates the in-vitro and in-vivo function of endothelial progenitor cells from hypertensive patients, via regulation of TSP-1 and soluble guanylyl cyclases/cyclic guanosine monophosphate system.