Tissue engineering endeavors to create replacement tissues and restore function that may be lost through infection, trauma, and cancer. However, wide clinical application of engineered scaffolds has yet to come to fruition due to inadequate vascularization. Here, we fabricate hydrogel constructs using Pluronic(®) F127 as a sacrificial microfiber, creating microchannels within biocompatible, biodegradable type I collagen matrices. Microchannels were seeded with human umbilical vein endothelial cells (HUVEC) or HUVEC and human aortic smooth muscle cells (HASMC) in co-culture, generating constructs with an internal endothelialized microchannel. Histological analysis demonstrated HASMC/HUVEC-seeded constructs with a confluent lining after 7 days with preservation and further maturation of the lining after 14 days. Immunohistochemical staining demonstrated von Willebrand factor and CD31(+) endothelial cells along the luminal surface (neointima) and alpha-smooth muscle actin expressing smooth muscle cells in the subendothelial plane (neomedia). Additionally, the deposition of extracellular matrix (ECM) components, heparan sulfate and basal lamina collagen IV were detected after 14 days of culture. HUVEC-only- and HASMC/HUVEC-seeded microchannel-containing constructs were microsurgically anastomosed to rat femoral artery and vein and perfused, in vivo. Both HUVEC only and HUVEC/HAMSC-seeded constructs withstood physiologic perfusion pressures while their channels maintained their internal infrastructure. In conclusion, we have synthesized and performed microvascular anastomosis of tissue-engineered hydrogel constructs. This represents a significant advancement toward the generation of vascularized tissues and brings us closer to the fabrication of more complex tissues and solid organs for clinical application.