Blood vessel growth and regression in human endometrium are regulated throughout the menstrual cycle. We sought a direct role of ovarian steroids on human endometrial endothelial cell (HEEC) proliferation and vascularization. To investigate the HEEC angiogenicity of sex steroids, we developed a reliable method for the isolation of HEEC, which allowed us to investigate the angiogenic effects of sex steroids using immunohistochemistry, immunocytochemistry, Western blot, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt proliferation, and vascular tube formation analyses. We were able to obtain 95–99% pure HEEC with our isolation technique. HEEC expressed predominantly estrogen receptor β, minimally expressed estrogen receptor α, and but did not express progesterone (P₄) receptors A and B in vivo and in vitro. Estradiol (E₂; 10⁻¹⁰–10⁻⁸m) and P₄ (10⁻¹²–10⁻⁸m), alone or in combination, induced HEEC proliferation compared with control values after 48 h of treatment (P < 0.05). Furthermore, after 8 d of treatment, there were significantly more angiogenic patterns in E₂ (10⁻⁸m), P₄ (10⁻¹⁰m), and E₂ plus P₄ (10⁻⁸ and 10⁻¹⁰m) treatment groups compared with the control group (angiogenic scores, 2.95 ± 0.16, 3.26 ± 0.16, 3.06 ± 0.17, and 1.93 ± 0.15, respectively; P < 0.01). In conclusion, our results suggest that there are direct effects of E₂ and P₄ on HEEC and provide a new understanding of the physiological role of sex steroids in the regulation of endometrial events such as angiogenesis.