Saponins, the primary phytochemicals contributing to the health properties of G. pentaphyllum were frequently studied. However, compounds responsible for its bioactivities were still poorly understood. The saponin-rich fraction (GPMS), 3-O-[2G-(E)-Coumaroyl-3G-O-beta-D-glucosyl-3R-O-beta-D-glucosylrutinoside] (KCGG), gypenoside XLVI and gypenoside L were obtained by purification of G. pentaphyllum. The compounds were examined and compared with GPMS for their inhibitory effects on LPS-induced nitric oxide (NO) production. GPMS and KCGG differed in their inhibitory capacities against pro inflammatory cytokines secretion. GPMS exhibited strong inhibition on inducible nitric oxide synthase (iNOS) and interleukin-6 (IL-6) mRNA expression but weak inhibition on tumor necrosis factor-alpha (TNF-alpha) and interleukin-1 beta mRNA expression. KCGG was better at inhibiting iNOS, IL-6, TNF-alpha, and cyclooxygenase-2 (COX-2) mRNA expression. GPMS showed similar inhibitory potency on mitogen-activated protein kinase phosphorylation and nuclear factor-kappa B (NF-kappa B) activation, as evidenced by their regulatory effects on LPS-induced P65 phosphorylation, NF-kappa B nuclear translocation, I kappa B alpha phosphorylation and degradation, I kappa K alpha/beta phosphorylation, c-Jun N-terminal kinase phosphorylation, P38 phosphorylation, and COX-2 expression. KCGG was more powerful in inhibiting the NF-kappa B pathway, suggesting that KCGG might be used in the management of inflammatory-associated diseases in which NF-kappa B played pivotal roles. Furthermore, KCGG might be mainly responsible for the predominant effects of GPMS.