Vitamin E gamma-tocotrienol (γTE) is known to have anticancer effects, but mechanisms underlying these actions are not clear. Here using liquid chromatography tandem mass spectrometry, we show that γTE induced marked changes of sphingolipids including rapid elevation of dihydrosphingosine and dihydroceramides (dhCers) in various types of cancer cells. The elevation of dihydrosphingolipids coincided with increased cellular stress, as indicated by JNK phosphorylation, and was prior to any sign of induction of apoptosis. Chemically blocking de novo synthesis of sphingolipids partially counteracted γTE-induced apoptosis and autophagy. Experiments using 13C3, 15N-labeled l-serine together with enzyme assays indicate that γTE inhibited cellular dihydroceramide desaturase (DEGS) activity without affecting its protein expression or de novo synthesis of sphingolipids. Unlike the effect on dhCers, γTE decreased ceramides (Cers) after 8-h treatment but increased C18:0-Cer and C16:0-Cer after 16 and 24 h, respectively. The increase of Cers coincides with γTE-induced apoptosis and autophagy. Since γTE inhibits DEGS and decreases de novo Cer synthesis, elevation of Cers during prolonged γTE treatment is likely caused by sphingomeylinase-mediated hydrolysis of sphingomyelin. This idea is supported by the observation that an acid sphingomeylinase inhibitor partially reversed γTE-induced cell death. Our study demonstrates that γTE altered sphingolipid metabolism by inhibiting DEGS activity and possibly by activating SM hydrolysis during prolonged treatment in cancer cells.