Megastigmane glycosides ( 1– 5) together with seven ( 6– 12) related known compounds were isolated from the whole plants of Gynostemma pentaphyllum. The structures were elucidated by means of spectroscopic methods, including 2D NMR, HR-ESIMS, and circular dichroism (CD), as well as chemical transformations to be (3 R, 4 R, 5 S, 6 S, 7 E)-3,4,6-trihydroxymegastigmane-7-en-9-one-3- O- β- d-glucopyranoside (gynostemoside A, 1), (3 S, 4 S, 5 R, 6 R, 7 E, 9 R)-3,4,6,9-tetrahydroxymegastigmane-7-en-3- O- β- d-glucopyranoside (gynostemoside B, 2), (3 S, 4 S, 5 S, 6 S, 7 E, 9 R)-3,4,9-trihydroxymegastigmane-7-en-9- O- β- d-glucopyranoside (gynostemoside C, 3), (3 S, 4 S, 5 S, 6 S, 7 E, 9 R)-3,4,9-trihydroxymegastigmane-7-en-3- O- β- d-glucopyranoside (gynostemoside D, 4), and (3 S, 4 S, 5 S, 6 S, 7 E, 9 R)-3,4,9-trihydroxymegastigmane-7-en-4- O- β- d-glucopyranoside (gynostemoside E, 5), respectively.