Oral propranolol (PRN) has recently been shown to be highly effective for infantile hemangiomas (IHs), and is currently recommended as the first-line treatment of complicated IHs. However, no pharmacokinetic parameters have ever been reported for infants who received PRN treatment for IHs, and the therapeutic mechanism(s) still remain unclear. In this study, we found that plasma PRN concentration was affected by the frequency of administration of PRN. A single daily administration of PRN (1 mg/kg/d) resulted in an early elevation of plasma PRN compared to administration of the same dose divided into two times daily. In contrast, administration of PRN twice per day resulted in a more prolonged expression at a later time-point. The study also demonstrated that application of PRN at a normal concentration equivalent to plasma concentration did not inhibit proliferation or promote apoptosis of hemangioma derived stem cells (HemSCs) isolated from IH patients. PRN suppressed expression of angiogenic factors VEGF and bFGF in HemSCs in vitro. Morphological, histological and immunohistological improvement were observed in vivo using murine IH model in which HemSCs pre-treated with PRN were implanted into BALB/c-nu mice. In the pre-treated HemSC grafts, mean micro-vessel density (MVD) significantly decreased and protein levels of VEGF markedly decreased, while bFGF was still detectable. The results suggested PRN inhibited angiogenesis via down-regulating the expression of vascular endothelial growth factor in hemangioma derived stem cell. These findings provide critical insight into the pharmacokinetics and potential mechanisms of PRN action on IH.