The anti-aging activity of marine macroalgae Gracilaria lemaneiformis polysaccharide (GP) on Caenorhabditis elegans was evaluated by observing the lifespan, reproduction, pharyngeal pumping and stress response of worms. Moreover, quantitative fluorescence of polyglutamic acid and nuclear localization of DAF-16 were observed. The results showed that GP treatment enhanced the mean lifespan by over 16.47% and significantly increased the reproduction duration of worm in the high dose group (1000 μg/mL). GP exhibited little potent effects under the thermotolerance and oxidative stress. The number of polyglutamic acid aggregates in three dosage groups decreased by 24.82%, 32.08% and 30.93% (p < 0.05) compared to the control. The middle dose group strongly induced DAF-16 nuclear translocation over intermediate and cytosolic localizations compared to the control (p < 0.001). It was inferred that GP extended the adult lifespan of wild-type and polyQ nematodes through the insulin pathway DAF-16.