Ascarosides are small-molecule signals that play a central role in C. elegans biology, including dauer formation, aging, and social behaviors, but many aspects of their biosynthesis remain unknown. Using automated 2D NMRbased comparative metabolomics, we identified ascaroside ethanolamides as shunt metabolites in C. elegans mutants of daf-22, a gene with homology to mammalian 3-ketoacyl-CoA thiolases predicted to function in conserved peroxisomal lipid β oxidation. Two groups of ethanolamides feature β-keto functionalization confirming the predicted role of daf-22 in ascaroside biosynthesis, whereas α-methyl substitution points to unexpected inclusion of methylmalonate at a late stage in the biosynthesis of long-chain fatty acids in C. elegans. We show that ascaroside ethanolamide formation in response to defects in daf-22 and other peroxisomal genes is associated with severe depletion of endocannabinoid pools. These results indicate unexpected interaction between peroxisomal lipid β-oxidation and the biosynthesis of endocannabinoids, which are major regulators of lifespan in C. elegans. Our study demonstrates the utility of unbiased comparative metabolomics for investigating biochemical networks in metazoans.