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Biologic function and the mechanical performance of vascular grafting materials are important predictors of graft patency. As such, "functional" materials that improve biologic integration and function have become increasingly sought after. An important alternative to synthetic materials is the use of biomaterials derived from ex vivo tissues that retain significant biologic and mechanical function. Unfortunately, inconsistent mechanical properties that result from tedious, time consuming, manual dissection methods have reduced the potential usefulness of many of these materials. We describe the preparation of the human umbilical vein (HUV) for use as an acellular, three-dimensional, vascular scaffold using a novel, automated dissection methodology. The goal of this investigation was to determine the effectiveness of the autodissection methodology to yield an ex vivo biomaterial with improved uniformity and reduced variance. Mechanical properties, including burst pressure, compliance, uniaxial tension testing, and suture holding capacity, were assessed to determine the suitability of the HUV scaffold for vascular tissue engineering applications. The automated methodology results in a tubular scaffold with significantly reduced sample to sample variation, requiring significantly less time to excise the vein from the umbilical cord than manual dissection methods. Short-term analysis of the interactions between primary human vascular smooth muscle cells and fibroblasts HUV scaffold have shown an excellent potential for cellular integration by native cellular remodeling processes. Our work has shown that the HUV scaffold is mechanically sound, uniform, and maintains its biphasic stress-strain relationship throughout tissue processing. By maintaining the mechanical properties of the native blood vessels, in concert with promising cellular interactions, the HUV scaffold may lead to improved grafts for vascular reconstructive surgeries.