Objectives: Berberine (BR) has a beneficial effect on endothelial function by increasing nitric oxide (NO), as NO plays a pivotal role in the regulation of endothelial progenitor cell (EPC) mobilization and function. The aim of the present study was to investigate whether BR-induced upregulation of circulating EPCs is related to NO production in healthy subjects. Methods: Twenty volunteers were recruited and received 400 mg of BR 3 times a day for 30 days. We assessed the number of EPC colony-forming units (EPC-CFUs), as well as the proliferative, adhesive and migratory activities of circulating EPCs before and after the 30-day BR therapy. The level of plasma NO was also measured before and after the 30-day BR therapy. Results: After 30 days of BR therapy, the number of EPC-CFUs was increased and the function of EPCs, including proliferation, adhesion and migration, was augmented. In parallel, BR therapy enhanced the plasma NO level. There was a significant linear regression relationship between the enhanced plasma NO level and the increased number and function of circulating EPCs. Conclusions: BR-induced upregulation of the number and function of circulating EPCs in healthy subjects is related to NO production.