Portulaca oleracea L. (PO), with abundant natural bioactive phytochemicals, exhibits potential bioactivities and pharmacological activities. However, the mechanisms of action of PO on anti-aging effect remain unclear. In this study, the ethyl acetate fractional extract from PO (PO-EA) was obtained by fractionation of solvent extractions, and its effect on lifespan was assessed using the Caenorhabditis elegans (C. elegans). Results showed that PO-EA could significantly increase the lifespan of C. elegans by 5.31, 12.67, and 16.47% at the doses of 250, 500, and 1,000 μg/mL, respectively. Moreover, PO-EA significantly promoted the mobility of C. elegans without obvious side effects such as changing body length or decreasing fecundity of the nematodes. Further study demonstrated that PO-EA could enhance the stress resistance in C. elegans via improving the activities of superoxide dismutase and catalase, and diminishing the contents of reactive oxygen species and malondialdehyde. The gene expression of daf-12, daf-16, sod-3, skn-1, cat-1, mev-1, akt-1, and sek-1 were upregulated in C. elegans after administrated by PO-EA. This study indicated that PO-EA plays a vital role in extending lifespan and healthspan in C. elegans, and the underlying mechanism of action might be attributed to Insulin/IGF-1-like signaling pathways. Therefore, PO-EA could be served as a potential candidate for anti-aging functional food.