Tao-Hong-Si-Wu decoction (TSD) is a famous traditional Chinese medicine (TCM) and widely used for ischemic disease in China. TSD medicated serum was prepared after oral administration of TSD (1.6 g/kg) twice a day for 3 days in rats. TSD medicated serum induced human umbilical vein endothelial cells (HUVECs) proliferation, VEGF secretion, and nitric oxide (NO) production. These promoted effects of TSD were partly inhibited by treatment with PI3K inhibitor (LY294002) or eNOS inhibitor (L-NAME), respectively, and completely inhibited by treatment with LY294002 and L-NAME simultaneously. Western blot analysis findings further indicated that TSD medicated serum upregulated p-Akt and p-eNOS expressions, which were significantly inhibited by LY294002 or L-NAME and completely inhibited by both LY294002 and L-NAME; these results indicated that TSD medicated serum induced HUVECs VEGF expression via PI3K/Akt-eNOS signaling. TSD medicated serum contains hydroxysafflor yellow A, ferulic acid, and ligustilide detected by UPLC with standards, so these effect of TSD medicated serum may be associated with these three active compounds absorbed in serum.