LC-MS-guided phytochemical isolation of malonylginsenosides, featuring neutral elimination of CO and CHO by the negative mode collision-induced dissociation, from the flower buds of Panax ginseng led to the isolation of 19 malonyl-substituted triterpenoid saponins. They include 15 new malonylginsenosides, malonylfloralginsenosides-Re-Re (1-3), -Rb and -Rb (4, 5), -Rd-Rd (6-11), and -Rc-Rc (12-15), and the known m-Rb, m-Rc, m-Rb, and m-Rd (16-19). Compound 11 represents the first dimalonyl saponin isolated from the Panax genus, while 2-4, 9, and 10 are five ginsenosides with single malonylation at the C-20 sugar chain. The antidiabetic activities of nine of these malonyl-substituted ginsenosides (1, 3, 4, 8, 13, and 16-19) and five of the corresponding non-malonyl ginsenosides (Re, Rb, Rb, Rc, and Rd) were evaluated by L6 myotubes' glucose consumption and AMPKα2β1γ1 activation. Ginsenoside Rb, 1, and 18 promoted glucose consumption of differentiated L6 myotubes, while ginsenosides Rb, Rb, and Rd and the malonylginsenosides 4, 8, 13, 16, 17, and 19 activated AMPKα2β1γ1 (EC: 0.0168-2.8 μM, fold: 1.7-4.7).