Jiaogulan (Gynostemma pentaphyllum) has been widely used as a herbal tea, dietary supplement, and vegetable in Asian countries. In this study, six new gypenosides (1–6) were isolated from the aerial parts of G. pentaphyllum. Their molecular structures were elucidated through spectroscopic analysis and acid hydrolysis. Gypenosides 1 and 2 represented the first example of a dammar-21-O- glucopyranoside without any unsaturated functional group and a dammar-3-O-glucopyranosyl-25-O-glucopyranoside without any cyclization in the side chain, respectively. In addition, gypenosides 5 and 6 exhibited the first example of a 24-hydroperoxy-19-oxo-dammarane triterpenoid and 19-oxo-dammar-21-O-glucopyranoside with a saturated side chain, respectively. Gypenoside 5 was found to possess protein tyrosine phosphatase 1B inhibitory activity, with an IC₅₀ value of 8.2 ± 0.9 μM, and moderate cytotoxicity against human breast cancer cells MCF7, MCF7/ADR, and MDA-MB-231, with IC₅₀ values ranging from 10.5 ± 1.4 to 14.2 ± 2.6 μM. The outcome of the study provided crucial information regarding the structural diversity and health benefits of gypenosides.