Mechanisms by which autophagy promotes cell survival or death are unclear. We provide evidence that C₁₈-pyridinium ceramide treatment or endogenous C₁₈-ceramide generation by ceramide synthase 1 (CerS1) expression mediates autophagic cell death, independent of apoptosis in human cancer cells. C₁₈-ceramide–induced lethal autophagy was regulated via microtubule-associated protein 1 light chain 3 β-lipidation, forming LC3B-II, and selective targeting of mitochondria by LC3B-II–containing autophagolysosomes (mitophagy) through direct interaction between ceramide and LC3B-II upon Drp1-dependent mitochondrial fission, leading to inhibition of mitochondrial function and oxygen consumption. Accordingly, expression of mutant LC3B with impaired ceramide binding, as predicted by molecular modeling, prevented CerS1-mediated mitochondrial targeting, recovering oxygen consumption. Moreover, knockdown of CerS1 abrogated sodium selenite–induced mitophagy, and stable LC3B knockdown protected against CerS1- and C₁₈-ceramide–dependent mitophagy and blocked tumor suppression in vivo. Thus, these data suggest a new receptor function of ceramide for anchoring LC3B-II autophagolysosomes to mitochondrial membranes, defining a key mechanism for the induction of lethal mitophagy.