The anti-cancer, vision-improving, and reproduction-enhancing effects of goji berry have been generally recognized, but its role in anti-aging is rarely studied in depth. Therefore, two widely-circulated goji berries, Lycium ruthenicum Murr. (LRM) and Lycium Barbarum. L (LB), were selected to explore their effects on extending lifespan and enhancing defense against extrinsic stress and to uncover the mechanism of action through genetic study. The results showed that supplementation with high-dose LRM (10 mg mL-1) and LB (100 mg mL-1) extracts significantly extended the lifespan of Caenorhabditis elegans (C. elegans) by 25.19% and 51.38%, respectively, accompanied by the improved stress tolerance of C. elegans to paraquat-induced oxidation, UV-B irradiation and heat shock. Furthermore, LRM and LB extracts remarkably enhanced the activities of antioxidant enzymes including SOD and CAT in C. elegans, while notably decreased the lipofuscin level. Further genetic research demonstrated that the expression levels of key genes daf-16, sod-2, sod-3, sir-2.1 and hsp-16.2 in C. elegans were up-regulated by the intervention with LRM and LB, while that of the age-1 level was down-regulated. Moreover, the daf-16 (mu86) I, sir-2.1 (ok434) IV and hsf-1 (sy441) I mutants reversed the longevity effect brought about by LRM or LB, which confirmed that these genes were required in goji berry-mediated lifespan extension. Therefore, we conclude that HSF-1 and SIR-2.1 act collaboratively with the insulin/IGF signaling pathway (IIS) in a daf-16-independent mode. The present study indicated goji berry as a potential functional food to alleviate the symptoms of aging.