<p>Background: Vascular Endothelial Growth Factor (VEGF) is a coordinate regulator of physiological angiogenesis during embryogenesis, skeletal growth and reproductive functions. There are several types of VEGF, including VEGF₁₆₅. VEGFs stimulate endothelial cell growth, angiogenesis, and capillary permeability. Low induction temperature is a major factor for expression of the recombinant VEGF₁₆₅ in soluble form. The purpose of this study was cloning and optimization of soluble vascular endothelial growth factor165 expression in <em>Escherichia coli (E. coli)</em>.<br /> Methods: In this study, total RNA of HeLa cell [cervix epithelium] was extracted. The VEGF₁₆₅ gene was amplified by reverse transcription polymerase chain reaction (RT-PCR), and then VEGF₁₆₅ was subcloned into prokaryotic expression vectors pET-32a(+) and transformed into BL21 (DE3) <em>E. coli</em> strain. VEGF₁₆₅ expression was optimized by fine adjustments such as induction time and incubation temperature. VEGF₁₆₅ was analyzed by DNA sequencing prior to expression and the protein was further characterized by SDS-PAGE and immunoblotting using His&bull;tag specific polyclonal antibody.<br /> Results: Our results demonstrated that VEGF₁₆₅ was successfully cloned and expressed in pET-32a(+) vector. Optimization of the expression procedure showed that, induction by 1 <em>mM</em> IPTG at OD600=0.7 and overnight incubation at 22^o<em>C</em> resulted in the highest expression levels of soluble VEGF₁₆₅.<br /> Conclusion: In this study, the expression of VEGF₁₆₅ in a high soluble level was successfully cloned and optimized.</p>