The present work investigates the effects of hydroxyl end groups on the electrospinning behavior of wheat gluten protein. We focus upon the impact of both a low molecular weight additive (a star-branched polyol: ethoxylated trimethylolpropane) and a high molecular weight additive (a synthetic biodegradable polymer: poly(vinyl alcohol) on the ability to form electrospun fibers from wheat gluten. The presence of the star-branched polyol in the system appears to impede the formation of molecular entanglements and intermolecular disulfide bridges required for fiber formation, while the addition of poly(vinyl alcohol) (PVOH) to the system leads to a significant increase in the overall number of physical chain entanglements required for fiber formation. The mechanical testing data support the overriding importance of physical chain entanglements as larger amounts of PVOH are combined with wheat gluten. The tensile strength of the wheat gluten-based electrospun fibrous mats, which, increased by an order of magnitude upon adding 13% (w/w) PVOH to the electrospinning solution, resulted in a rapid increase in the measured toughness at 26% (w/w) PVOH. The corresponding scanning electron micrographs reveal that the addition of PVOH to the gluten system results in the formation of flat, ribbonlike fibers. Dough mixograms were also collected to further elucidate the effect of additional hydroxyl groups on the interactions between the gluten protein chains. Evidence reveals that in the high-concentration, doughy regime, a small fraction of the protein-water interactions are replaced by favorable polyol-protein interactions; however, in the dilute-concentration regime, these favorable polyol-protein interactions appear to develop at the expense of disulfide bond formation, a requirement for obtaining fibers via electrospinning.