Background: Notoginsenoside R2 (NGR2) is an important active saponin molecule of Panax Notoginseng (PN), but its effect on regulating angiogenesis is unclear. Here, we investigated the role of NGR2 in angiogenesis in vivo and in vitro. Methods: NGR2 was administered to rats by intragastric administration for 7 days. The colonic histopathology and microvessel density (MVD) were observed and evaluated under an inverted microscope. The colonic mucosal permeability (MP) and vascular permeability (VP) were evaluated by measuring the transmittance of FD-4 and the vascular leakage of Evans blue, respectively. The serum IL-2, TNF-alpha, IL-4, IL-10, VEGFA165, and VEGFA121 levels were detected with ELISA. In vitro, pHUVECs were cultured and treated with NGR2. The mechanism of NGR2 in angiogenesis was assessed by VEGFA165, LY294002, silencing, and overexpression plasmids of Rap1 GAP in vitro. The cell viability, cell proliferation, tube formation, and concentration of intracellular pyruvate and lactic acid of pHUVECs were measured after 24 h. The expression of proteins was detected with western blotting. Results: NGR2 could significantly induce inflammatory injuries in the colonic mucosa and microvessels. Both MP and VP in rats treated with NGR2 increased in proportion to the serum VEGFA165 and VEGFA121 level, the ratio of VEGFA165/VEGFA121, and the concentration of intracellular pyruvate and lactic acid. In vitro, NGR2 reduced cell viability, proliferation, and tube formation, and enhanced the intracellular glycolysis of pHUVECs. Furthermore, the cell viability, proliferation, and tube formation of pHUVECs were inhibited by NGR2 via blocking the Rap1GAP/PI3K/Akt signaling pathway. Conclusions: NGR2 could induce colonic mucosal microvascular injuries and promoted the intracellular glycolysis of pHUVECs via blocking the Rap1GAP/PI3K/Akt signaling pathway.