Probiotics are bacteria among the intestinal flora that are beneficial for human health. Bifidobacterium longum (BL) is a prototypical probiotic that is widely used in yogurt making, supplements and others. Although various physiological effects of BL have been reported, those associated with longevity and anti-ageing still remain elusive. Here we aimed to elucidate the physiological effects of killed BL (BR-108) on stress tolerance and longevity of Caenorhabditis elegans and their mechanisms. Worms fed killed BL in addition to Escherichia coli (OP50) displayed reduced body length in a BL dose-dependent manner. When compared with those fed E. coli alone, these worms had a higher survival rate following heat stress at 35°C and hydrogen peroxide-induced oxidative stress. A general decrease in motility was observed over time in all worms; however, killed BL-fed ageing worms displayed increased movement and longer life span than those fed E. coli alone. However, the longevity effect was suppressed in sir-2.1, daf-16 and skn-1-deficient worms. Killed BL induced DAF-16 nuclear localisation and increased the expression of the DAF-16 target gene hsp-12.6. These results revealed that the physiological effects of killed BL in C. elegans were mediated by DAF-16 activation. These findings contradict previous observations with different Bifidobacterium and Lactobacillus strains, which showed the role for SKN-1 independently of DAF-16.