Nanoscale structures of whey protein isolate (WPI) pretreated by microbial transglutaminase (mTGase) and subsequent heating were studied in this work and were correlated to zeta-potential, surface hydrophobicity, thermal denaturation properties, and macroscopic turbidity and viscosity. Dispersions of 5% w/v WPI were pretreated by individual or sequential steps of preheating at 80 °C for 15 min and mTGase, used at 2.0-10.2 U/g WPI for 1-15 h, before adjustment of the pH to 7.0 and to 0-100 mM NaCl for heating at 80 °C for 15 and 90 min. The zeta potential and surface hydrophobicity of WPI increased after all pretreatment steps. Preheating increased cross-linking reactivity of WPI by mTGase, corresponding to significantly increased denaturation temperature. Particle size analysis and atomic force microscopy revealed that structures of sequentially pretreated WPI remained stable after heating at 100 mM NaCl, corresponding to transparent dispersions. Conversely, WPI pretreated by one step aggregated at only 100 mM NaCl and resulted in turbid dispersions. Besides reporting a practical approach to produce transparent beverages, nanoscale phenomena in the present study are important for understanding whey protein structures in relevant applications.