The beneficial effects of garlic (Allium sativum) consumption in treating human diseases have been reported worldwide over a long period of human history. The strong antioxidant effect of garlic extract (GE) has also recently been claimed to prevent cancer, thrombus formation, cardiovascular disease and some age-related maladies. Using Caenorhabditis elegans as a model organism, aqueous GE was herein shown to increase the expression of longevity-related FOXO transcription factor daf-16 and extend lifespan by 20%. By employing microarray and proteomics analysis on C. elegans treated with aqueous GE, we have systematically mapped 229 genes and 46 proteins with differential expression profiles, which included many metabolic enzymes and yolky egg vitellogenins. To investigate the garlic components functionally involved in longevity, an integrated metabolo-proteomics approach was employed to identify metabolites and protein components associated with treatment of aqueous GE. Among potential lifespan-promoting substances, mannose-binding lectin and N-acetylcysteine were found to increase daf-16 expression. Our study points to the fact that the lifespan-promoting effect of aqueous GE may entail the DAF-16-mediated signaling pathway. The result also highlights the utility of metabolo-proteomics for unraveling the complexity and intricacy involved in the metabolism of natural products in vivo.