Paeonia lactiflora Pall., a plant of the genus Paeonia, which includes Radix paeoniae Rubra and R. paeoniae Alba has a long history in the traditional Chinese medicine for the management of a variety of ailments. These multiple therapeutic activities have been associated with paeoniflorin, an active component of the roots of R. paeoniaes. Herein, we have evaluated the effect of paeoniflorin on the survival, exercise capacity, antioxidant enzyme activity, and expression of genes hsp-16.2 and daf-16 in C. elegans, a soil-borne free-living nematode, under heat stress at 35°C. Paeoniflorin increased the ability to exercise, survival rate under heat stress, and in vivo activity of antioxidant enzymes significantly. Furthermore, treatment of transgenic C. elegans nematode (TJ375) expressing a jellyfish green fluorescent protein-tagged inducible small heat-shock protein gene with paeoniflorin led to a significant suppression of gene expression. After 2 h of 35°C heat shock exposure there was an enhanced translocation of daf-16 into the nucleus in paeoniflorin-treated nematodes. Similarly, the results of real-time polymerase chain reaction study showed that paeoniflorin promoted expression of daf-16 mRNA levels and reduced the transcription of HSP-16.2 gene under heat stress. In summary, these results show that paeoniflorin has a protective effect on heat stress in C. elegans.