The term probiotic refers to bacteria that provide a beneficial effect to the host. Limosilactobacillus reuteri (Lactobacillus reuteri) is a probiotic isolated from human breast milk. Although L. reuteri has antimicrobial and anti-inflammatory activities occasionally linked to anti-aging effects, there are no reports of the effects of L. reuteri on longevity. This study evaluated the anti-aging effects of L. reuteri on the lifespan and physiology of Drosophila melanogaster. L. reuteri increased the mean lifespan of fruit flies significantly without reducing the reproductive output, food intake, or locomotor activity. Furthermore, the data suggested that the longevity effect of L. reuteri is mediated by the reduction of the insulin/IGF-1 signaling pathway and the action of reuterin, an antimicrobial compound produced by L. reuteri. These results show that L. reuteri can be used as a probiotic that acts as a dietary restriction mimetic with anti-aging effects.