Electrospun fibers with an average fiber diameter in the nanometer range were prepared from soy protein isolate to develop scaffolds for tissue engineering applications. Poly(ethylene oxide) was added to facilitate fiber formation. The influence of processing parameters such as applied voltage, soy protein isolate and poly(ethylene oxide) concentrations, and poly(ethylene oxide) molecular weight on electrospun fiber morphology was investigated. Resulting soy protein isolate/poly(ethylene oxide) mats were carbodiimide crosslinked to increase construct robustness. Mechanical properties and in vitro biocompatibility of crosslinked electrospun scaffolds were evaluated. Soy protein isolate/poly(ethylene oxide) fiber diameters ranged between 50 and 270 nm depending on both electrospinning and solution parameters. The Young's modulus for 7% soy protein isolate/3% poly(ethylene oxide) and 12% soy protein isolate/3% poly(ethylene oxide) electrospun scaffolds were 75 and 252 kPa, respectively. Human mesenchymal stem cell studies showed successful cell adhesion and proliferation on the soy protein isolate/poly(ethylene oxide) fibers. The structural and biological properties of these soy protein isolate electrospun scaffolds suggest their potential applications in tissue engineering.