Endothelial oxidative stress induces cellular activation and sometimes death. Endothelial death can occur via necrosis or apoptosis. Understanding the mechanisms involved in cellular activation and death may lead to therapeutics designed to increase death or preserve cellular function. In the present study, brief periods of anoxia (3 h) followed by varying lengths of reoxygenation (0-5 h) lead to a time-dependent increase in human umbilical vein endothelial cell (HUVEC) caspase activity. Furthermore, ROCK-1 cleavage, which is dependent on caspase-3 activity, was also increased in cells undergoing oxidative stress compared with normoxic cells. Microarray data demonstrated that glucosylceramide synthase (GCS; glucosylceramide transferase), but not acid sphingomyelinase, was modulated by anoxia and reoxygenation. We confirmed that GCS mRNA and protein expression were significantly decreased in a time-dependent fashion following oxidative stress by real-time polymerase chain reaction and Western blot, respectively. Treatment of normoxic cells with the GCS-specific inhibitor, D,L-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP), increased caspase activity to the same degree as cells undergoing oxidative stress. Fumonisin B1, the N-acyl-sphinganine dehydrogenase (e.g., ceramide synthase) inhibitor significantly attenuated caspase activity in HUVECs undergoing oxidative stress. These data suggest that alterations in GCS expression following brief periods of oxidative stress in human endothelial cells lead to increased caspase activity.