In organisms, high glucose can cause several aspects of toxicity, including the lifespan reduction. Paeoniflorin is the major component of Paeoniaceae plants. Nevertheless, the possible effect of paeoniflorin to suppress high glucose toxicity in reducing lifespan and underlying mechanism are largely unclear. Thus, in this study, we examined the possible effect of paeoniflorin in suppressing high glucose (50 mM)-induced lifespan reduction and the underlying mechanism inCaenorhabditis elegans. Administration with 16–64 mg/L paeoniflorin could prolong the lifespan in glucose treated nematodes. Accompanied with this beneficial effect, in glucose treated nematodes, expressions ofdaf-2encoding insulin receptor and its downstream kinase genes (age-1,akt-1, andakt-2) were decreased and expression ofdaf-16encoding FOXO transcriptional factor was increased by 16–64 mg/L paeoniflorin administration. Meanwhile, the effect of paeoniflorin in extending lifespan in glucose treated nematodes was enhanced by RNAi ofdaf-2,age-1,akt-1, andakt-2and inhibited by RNAi ofdaf-16. In glucose treated nematodes followed by paeoniflorin administration, the increased lifespan caused bydaf-2RNAi could be suppressed by RNAi ofdaf-16, suggesting that DAF-2 acted upstream of DAF-16 to regulate pharmacological effect of paeoniflorin. Moreover, in glucose treated nematodes followed by paeoniflorin administration, expression ofsod-3encoding mitochondrial Mn-SOD was inhibited bydaf-16RNAi, and the effect of paeoniflorin in extending lifespan in glucose treated nematodes could be suppressed bysod-3RNAi. Molecular docking analysis indicated the binding potential of paeoniflorin with DAF-2, AGE-1, AKT-1, and AKT-2. Therefore, our results demonstrated the beneficial effect of paeoniflorin administration in inhibiting glucose-induced lifespan reduction by suppressing signaling cascade of DAF-2-AGE-1-AKT-1/2-DAF-16-SOD-3 in insulin signaling pathway.