Caenorhabditis elegans is an important model organism for studying stress response mechanisms and identifying genetic pathways that influence longevity. The present study was designed to explore the in vivo-antioxidant potential and the probable mechanism of acid hydrolysates prepared from A. auricula polysaccharides (AAPHs-F) with the optimal acid hydrolysis conditions using Box-Behnken design, and C. elegans was used as a model organism. The effects of AAPHs-F on the locomotory behavior, lifespan, activities of antioxidant-related enzymes and levels of antioxidants in C. elegans were studied. In addition, the potential of AAPHs-F in up-regulating the expression of antioxidant-related genes in C. elegans, such as daf-16, skn-1, sod-1, sod-2 and sir-2.1, and the inhibition of cell apoptosis of C. elegans were also discussed. The results indicated that AAPHs-F could significantly increase the U-Turn frequency of nematodes, extend their lifespan, enhance antioxidant systems including superoxide dismutase (SOD) by 70.60%, catalase (CAT) by 73.45% and glutathione reductase (GR) by 258.68% (p < 0.01), increase the level of glutathione (GSH) by 110.22% (p < 0.01), and decrease the level of reactive oxygen species (ROS) and malondialdehyde (MDA) by 31.86% and 46.16% (p < 0.01), respectively. Quantitative real-time polymerase chain reaction (qRT-PCR) results showed that AAPHs-F could up-regulate mRNA expression levels of daf-16, skn-1, sir, sod-1 and sod-2 in wild-type C. elegans (>1.3 fold) when treated at a concentration of 0.1 mg mL-1 (p < 0.05 or p < 0.01). AAPHs-F was concluded to be heteropolysaccharides composed of mannose, glucose and galactose with a molar ratio of 12.7 : 3.25 : 1. The molecular weight of AAPHs-F was determined to be 885.37 Da. Furthermore, AAPHs-F is mainly formed of (1 → 3)-linked-α-d-glucopyranose, and carboxyl or acetamide is present in the molecule. In summary, our studies provide evidence that AAPHs-F helps improve the antioxidant defense system, and up-regulation of stress and longevity related genes suggests the possible involvement of these genes in the prevention of stress damage in C. elegans.