Endothelial cells (ECs) are exposed to cytotoxic reactive oxygen species and oxidation products of NO, yet they are characterized by low apoptotic rates and have an average life span of many years. EC exposure to flow has been shown to downregulate cell cycle-related genes and cause cytoskeletal rearrangement. We hypothesized that exposure to flow also causes molecular and physiological changes that induce antioxidant properties in ECs. We used cDNA array expression profiling and protein analysis to study the responses of human ECs exposed to flow in a hollow fiber apparatus or the same ECs grown under static conditions. Our results show that shear-induced synchronized expression of processes control oxidant production; these changes included upregulation of NADH-producing enzymes (Krebs cycle dehydrogenases and glyceraldehyde-3-phosphate dehydrogenase [GAPDH]) accompanied by simultaneous decrease in NADH-depleting pathways (e.g., lactate dehydrogenase [LDH]) and diminished production of lactate. Exposure to flow upregulated cytoskeletal genes. Our results suggest that, in addition to inhibition of cell cycle, exposure to flow influences ECs by controlling expression of enzymes involved in the generation of antioxidant intermediates and in adaptive control of cell shape. These changes may explain longevity and antioxidant efficiency of ECs and may provide insight in mechanisms leading to pathological conditions such as arteriosclerosis.