The present study was undertaken to investigate the role of vascular endothelial growth factor (VEGF) in luteal angiogenesis and the regulation of VEGF in the corpus luteum (CL) during mid-pregnancy in rats. Protein concentrations and mRNA levels of VEGF in the CL significantly increased from Day 9 to Day 12 and remained at the same level as Day 12 until Day 15. To study whether estradiol is involved in VEGF expression between Day 12 and Day 15, rats undergoing hypophysectomy-hysterectomy on Day 12 were treated with estradiol until Day 15. Protein concentrations and mRNA levels of VEGF in the CL were significantly decreased by hypophysectomy-hysterectomy, and this inhibitory effect was completely reversed by estradiol treatment. Changes in vascular density in the CL were parallel to those in VEGF expression. To examine whether the effect of estradiol is mediated by VEGF, anti-VEGF antibody was administered to hypophysectomized-hysterectomized rats simultaneously with estradiol. The recovery in the vascular density, CL weight, and serum progesterone concentration caused by estradiol was significantly inhibited by the anti-VEGF antibody treatment. In conclusion, the present study has demonstrated that VEGF contributes to luteal angiogenesis, CL development, and progesterone production during mid-pregnancy in rats and that luteal VEGF expression is increased by estradiol.