K-Ras must localize to the plasma membrane (PM) for biological activity. We show here that multiple acid sphingomyelinase (ASM) inhibitors, including tricyclic antidepressants, mislocalized phosphatidylserine (PtdSer) and K-RasG12V from the PM, resulting in abrogation of K-RasG12V signaling and potent, selective growth inhibition of mutant K-Ras-transformed cancer cells. Concordantly, in nude mice, the ASM inhibitor fendiline decreased the rate of growth of oncogenic K-Ras-expressing MiaPaCa-2 tumors but had no effect on the growth of the wild-type K-Ras-expressing BxPC-3 tumors. ASM inhibitors also inhibited activated LET-60 (a K-Ras ortholog) signaling in Caenorhabditis elegans , as evidenced by suppression of the induced multivulva phenotype. Using RNA interference against C. elegans genes encoding other enzymes in the sphingomyelin (SM) biosynthetic pathway, we identified 14 enzymes whose knockdown strongly or moderately suppressed the LET-60 multivulva phenotype. In mammalian cells, pharmacological agents that target these enzymes all depleted PtdSer from the PM and caused K-RasG12V mislocalization. These effects correlated with changes in SM levels or subcellular distribution. Selected compounds, including sphingosine kinase inhibitors, potently inhibited the proliferation of oncogenic K-Ras-expressing pancreatic cancer cells. In conclusion, these results show that normal SM metabolism is critical for K-Ras function, which may present therapeutic options for the treatment of K-Ras-driven cancers.