Nutrient transport remains a major limitation in the design of biomaterials. One approach to overcome this constraint is to incorporate features to induce angiogenesis-mediated microvasculature formation. Angiogenesis requires a temporal presentation of both pro- and anti-angiogenic factors to achieve stable vasculature, leading to increasingly complex biomaterial design scheme. The endometrium, the lining of the uterus and site of embryo implantation, exemplifies a non-pathological model of rapid growth, shedding, and re-growth of dense vascular networks regulated by the dynamic actions of estradiol and progesterone. In this study, we examined the individual and combined response of endometrial epithelial cells and human umbilical vein endothelial cells to exogenous estradiol within a three-dimensional collagen scaffold. While endothelial cells did not respond to exogenous estradiol, estradiol directly stimulated endometrial epithelial cell transduction pathways and resulted in dose-dependent increases in endogenous VEGF production. Co-culture experiments using conditioned media demonstrated estradiol stimulation of endometrial epithelial cells can induce functional changes in endothelial cells within the collagen biomaterial. We also report the effect of direct endometrial epithelial and endothelial co-culture as well as covalent immobilization of estradiol within the collagen biomaterial. These efforts establish the suitability of an endometrial-inspired model for promoting pro-angiogenic events within regenerative medicine applications. These results also suggest the potential for developing biomaterial-based models of the endometrium. Biotechnol. Bioeng. 2015;112: 2185-2194. (c) 2015 Wiley Periodicals, Inc.