Abstract: To investigate the effects of D-allulose (D-Alu) on the lifespan and antioxidant capacity of Drosophila melanogaster, D. melanogaster were fed with different concentrations (0, 1, 5, 10 mg/mL) of D-allulose, and experiments on lifespan, climbing, reproduction, food intake, intestinal homeostasis, and antioxidant enzyme activities were carried out. The results showed that the mean and maximum lifespan of D. melanogaster in the group with 5 mg/mL of D-allulose were prolonged by 18.86% and 25.77%, which were significantly higher than those in the control group (P<0.05). The exercise capacity of aged D. melanogaster was significantly improved, and the climbing index of D. melanogaster at 30 d of age with the addition of D-allulose 5 mg/mL was increased by 21.10% compared with the control group (P<0.01). The egg production of D. melanogaster in the group supplemented with D-allulose 1 mg/mL increased by 10.9% (P<0.05) and the developmental cycle of D. melanogaster was shortened by 7.81% (P<0.01) compared with the control group. In addition, the addition of D-allulose 5 mg/mL increased the intestinal length of 30-d-old D. melanogaster by 13.06% (P<0.01) compared with the control group, and the proportion of intestinal leakage was reduced by 4.06% (P<0.01) compared with the control group, which reduced the shortening of the intestinal tract and the increase in permeability caused by aging. In the antioxidant assay, the mean survival time of D. melanogaster under oxidative stress conditions was prolonged by 12.34% (P<0.05) in the group supplemented with D-allulose 5 mg/mL, and the activities of D. melanogaster's superoxide dismutase (SOD) Cu/Zn-SOD and Mn-SOD were increased by 13.49% (P<0.01) and 16.19% (P<0.05), catalase (CAT) activity increased by 32.21% (P<0.01), and malonaldehyde (MDA) content decreased by 20.75% (P<0.01) compared to the control. In conclusion, D-allulose can significantly improve the antioxidant capacity and prolong the healthy lifespan of D. melanogaster with potential anti-aging efficacy.