Vascular endothelial growth factor (VEGF) is an important regulator of neovascularization. Hypoxia inducible nitric oxide (NO) enhanced the expression of VEGF and thymosin beta-4 ($T{eta}4$), actin sequestering protein. Here, we investigated whether NO-mediated VEGF expression could be regulated by $T{eta}4$ expression in HeLa cervical cancer cells. Hypoxia inducible NO production and VEGF expression were reduced by small interference (si) RNA of $T{eta}4$. Hypoxia response element (HRE)-luciferase activity and VEGF expression were increased by the treatment with N-(${eta}$-D-Glucopyranosyl)-N2-acetyl-S-nitroso-D, L-penicillaminamide (SNAP-1), to generate NO, which was inhibited by the inhibition of $T{eta}4$ expression with $T{eta}4$-siRNA. In hypoxic condition, HRE-luciferase activity and VEGF expression were inhibited by the treatment with $N^G$-monomethyl-L-arginine (L-NMMA), an inhibitor to nitric oxide synthase (NOS), which is accompanied with a decrease in $T{eta}4$ expression. VEGF expression inhibited by L-NMMA treatment was restored by the transfection with pCMV-$T{eta}4$ plasmids for $T{eta}4$ overexpression. Taken together, these results suggest that $T{eta}4$ could be a regulator for the expression of VEGF via the maintenance of NOS activity.