Evidence for a regulatory role of the miR-34 family in senescence is growing. However, the exact role of miR-34 in aging in vivo remains unclear. Here, we report that a mir-34 loss-of-function mutation in Caenorhabditis elegans markedly delays the age-related physiological decline, extends lifespan, and increases resistance to heat and oxidative stress. We also found that RNAi against autophagy-related genes, atg4, bec-1, or atg9, significantly reversed the lifespan-extending effect of the mir-34 mutants. Furthermore, miR-34a inhibits Atg9A expression at the post-transcriptional level in vitro, and the miR-34a binding sequences in the 3'-UTR of Atg9A contributes to the modulation of Atg9A expression by miR-34a. Our results demonstrate that the C. elegans mir-34 mutation extends lifespan by enhancing autophagic flux in C. elegans, and that miR-34 represses autophagy by directly inhibiting the expression of the autophagy-related proteins Atg9 in mammalian cells.