Three analogs of the dihydroceramide desaturase inhibitor XM462 are reported. The compounds inhibit both dihydroceramide desaturase and acid ceramidase, but with different potencies depending on the N-acyl moiety. Other enzymes of sphingolipid metabolism, such as neutral ceramidase, acid sphingomyelinase, acid glucosylceramide hydrolase, sphingomyelin synthase and glucosylceramide synthase, are not affected. The effect on the sphingolipidome of the two best inhibitors, namely (R,E)-N-(1-hydroxy-4-(tridecylthio)but-3-en-2-yl)octanamide (RBM2-1B) and (R,E)-N-(1-hydroxy-4-(tridecylthio)but-3-en-2-yl)pivalamide (RBM2-1D), is in accordance with the results obtained in the enzyme assays. These two compounds reduce cell viability in A549 and HCT116 cell lines with similar potencies and both induced apoptotic cell death to similar levels than C8-Cer in HCT116 cells. The possible therapeutic implications of the activities of these compounds are discussed.