Objective To construct three-dimensional (3D) pre-vascularized microstructures and explore the promoting effect of human fibroblasts (HFs) on the sprout and migration of human umbilical vein endothelial cells (HUVECs) in 3D co-culture system. Methods HUVECs and HFs were cultured and the 3rd to 5th generation cells were selected for subsequent experiments. In 2D co-culture system, HFs were stained with PKH26 and the cell density was fixed, which co-cultured with HUVECs in different ratios (1∶4, 1∶1, 4∶1) and inoculation methods (HUVECs inoculation at 48 hours after HFs, direct mixed inoculation). Then the formation of vascular like structures was observed under fluorescence microscope. In 3D co-culture system, HUVECs and HFs were labeled with green fluorescent protein and red fluorescent protein by lentivirus transfection, respectively. They were inoculated on porous micro-carriers followed by dynamically culturing in rotating bottles to prepare HF, HUVEC, HF-EC, or HF&EC microstructures. The cell growth in microstructures was testing by low permeability crystal violet staining. Subsequently, the microstructures were embedded in fibrin gel and the cell growth and adhesion in HF and HUVEC microstructures were observed by laser confocal microscopy. Laser confocal microscope were also used to observe the sprouts of 4 kinds of microstructures, as well as the cell composition, the number and length of sprouts from HF-EC and HF&EC microstructures. HFs conditioned medium was prepared to observe its effect on sprouts of HUVEC microstructures with DMEM as control group. Results In 2D co-culture system, HFs pre-culturing was helpful to the formation and stability of vascular like structures, and the best effect was when the ratio of two kinds of cells was 1∶1. In 3D co-culture system, it was found that the cells grew well on micro-carriers and had the ability of pre-vascularization. HUVEC microstructures did not sprout, but HF, HF-EC, and HF&EC microstructures could which indicated a good vascularization ability. The HF-EC microstructures were superior to HF&EC microstructures in terms of sprouts length and number (P<0.05). The tubes sprouting from co-cultured group were composed of HFs and HUVECs, and HF microstructures migration preceded HUVEC microstructures always, and their migration trajectories were the same. HUVEC microstructures could sprout when cultured in HFs conditioned media. Conclusion HF-HUVEC pre-vascularized microstructures can be prepared by pre-culturing HFs before HUVECs and with the cell ratio at 1∶1 in a rotating bottle. In 3D co-culture system, HFs can promote and guide the sprout of HUVECs.