Alzheimer's disease (AD) is a common form of dementia and amyloid-β peptide (Aβ) aggregation is considered to be one of its main causes. Paeoniflorin has been previously shown to attenuate cognitive damage inflicted by exogenous Aβ protein. Using transgenic Caenorhabditis elegans models expressing human Aβ1-42, we demonstrate here that paeoniflorin can delay progressive paralysis caused by endogenous Aβ expression and reduce the amount of toxic Aβ oligomers in vivo, although it has no effect on Aβ aggregation in vitro. Paeoniflorin does not, however, affect the lifespan of either wild-type or AD-like nematodes, implying a mechanism independent of a general antiaging effect. We then demonstrate that paeoniflorin can reduce reactive oxygen species levels in C. elegans AD models, which may contribute to its in vivo suppression of Aβ toxicity. Moreover, paeoniflorin is shown to upregulate the expression of the small heat shock protein HSP-16.2 as it is capable of increasing the hsp-16.2 transcript level in wild-type as well as AD-like nematodes and enhancing the fluorescence intensity in hsp-16.2::GFP nematodes. Taken together, our findings demonstrate the underlying mechanisms of the protective effect of paeoniflorin against age-onset Aβ proteotoxicity, which are, in part, connected with oxidative and heat shock stress responses.