Although the modulated expression of Dicer is documented upon neoplastic transformation, little is known for the regulation of Dicer expression by environmental stimuli and its roles in the regulation of cellular functions in primary cells. In this study, we found that Dicer expression was downregulated upon serum withdrawal in human umbilical vein endothelial cells (HUVECs). Serum withdrawal induced time-dependent repression of Dicer expression, which was specifically rescued by vascular endothelial cell growth factor or sphingosine-1-phosphate. When Dicer expression was silenced by shRNA against Dicer, cells were more prone to apoptosis under serum withdrawal, while the rate of apoptosis was comparable to control cells in serum-containing condition. Real time PCR-based gene expression profiling identified several genes whose expression was modulated by Dicer silencing, including adhesion and matrix-related molecules, Caspase 3 and nitric oxide synthase (NOS) 3. Dicer silencing markedly impaired migratory functions without affecting cell adhesion, and repressed phosphorylation of FAK and PYK2 in adherent HUVECs. Dicer knockdown upregulated Caspase 3 and downregulated NOS3 expression, and serum withdrawal indeed increased Caspase 3, and decreased NOS3 expression. Further, overexpression of Dicer in HUVECs resulted in marked reduction in apoptosis upon serum withdrawal, and decreased Caspase 3 and increased NOS3 expression. Inhibition of NOS activity by N-nitro-L-arginine methyl ester abrogated the effect of Dicer overexpression to rescue the cells from serum withdrawal induced apoptosis. These results indicated that serum withdrawal decreases Dicer expression, leading to increased susceptibility to apoptosis through the regulation of Caspase 3 and NOS3 expression.