Abstract: Electrostatic interaction between enzyme molecule and leather is a critical factor influencing the mass transfer behavior of enzyme during the leather-making process. In this study, glutamic acid (GA) and sodium gluconate (SG) were selected as charge regulators to increase the electrostatic repulsion between protease and collagen fibers in pelt, thereby decreasing their affinity and subsequently increasing the mass transfer efficiency and improving the bating performance. The results demonstrated that, under the typical bating condition (pH 8.5), GA and SG effectively decreased the zeta potential of trypsin solution from −8.5 mV to −11.2 mV and −12.4 mV, respectively, while retaining comparable proteolytic activity. GA and SG exhibited excellent mass transfer enhancement effects, reducing the time required for complete penetration by 82.1% and 71.4%, respectively. After bating with GA or SG for 60 min, the collagen damage decreased by 0.3% and 11.6%, respectively; the ammonia nitrogen concentrations of the effluents reduced by 88.1% and 80.1%, respectively, compared to bating with ammonium sulfate, an enhancer reported in our previous study. These findings suggest that GA and SG can not only effectively enhance protease transfer and reduce collagen damage through the electrostatic regulation mechanism, but also significantly decrease the pollution load of wastewater, demonstrating a promising application prospect for replacing ammonium sulfate to achieve green and efficient bating.