Electrospinning is an increasingly popular technique to generate 3D fibrous tissue scaffolds that mimic the submicron sized fibers of extracellular matrices. A major drawback of electrospun scaffolds is the small interfibrillar pore size, which normally prevents cellular penetration in between fibers. In this study, we introduced a novel process, based on electrospinning, to manufacture a unique gradient porous fibrous (GPF) scaffold from soy protein isolate (SPI). The pore sizes in the GPF scaffolds gradually increase from one side of the scaffold to the other, ranging from 7.8 +/- 2.5 mu m in the small pore side, 21.4 +/- 10.3 mu m in the mid layer to 58.0 +/- 23.6 mu m in the large pore side. The smallest pores of the GPF scaffolds appeared to be somewhat larger than those in conventionally electrospun SPI scaffolds (4.2 +/- 1.3 mu m). Hydrated GPF scaffolds exhibited J-shaped stress-strain curves, reminiscent of those for soft biological scaffolds. Attachment, spreading, and proliferation of human dermal fibroblasts (HDFB) were supported on both the small and the large pore sides of the GPF scaffolds. Cultured HDFB and murine RAW 264.7 macrophages penetrated significantly deeper (98.7 +/- 24.2 mu m and 53.3 +/- 9.6 mu m, respectively) into the larger pores than when seeded onto the small pore side of GPF scaffolds (22.8 +/- 6.2 mu m and 25.7 +/- 7.3 mu m) and control SPI scaffolds. (11.3 +/- 3.8 mu m and 15.3 +/- 3.1 mu m). This study introduces a novel fabrication technique, which, by convergence of several biofabrication technologies, produces scaffolds with enhanced cellular penetration.