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The aim of this research was to study the feasibility of using Wheat Germ Protein Isolate (WGPI) and Xanthan Gum (XG) as egg substitutes in mayonnaise formulation. The mayonnaise prepared with different compositions of WGPI, egg, and XG by applying the optimal mixture design method and effect of this substitution on its stability, heat stability, viscosity, texture, physicochemical and sensory characteristics of mayonnaise were considered. In order to investigate the quality of mayonnaise during storage, stability, viscosity, pH, and acidity of the samples were determined during storage for 4 months. These parameters were in acceptable ranges, throughout the storage time, in all samples except for formulation 8 that contained 50% egg replacement with WGPI and without XG. The stability, viscosity, firmness, and cohesiveness increased with increasing the substitution levels of WGPI, although the appearance and color scores decreased. Generally, samples containing WGPI and XG, had acceptable quality in terms of investigated properties. However, the sensory characteristics of mayonnaise samples were decreased by increasing WGPI substitution. Mayonnaise with proper physicochemical and sensory properties was prepared by replacing %71.5 and %4.2 of egg by WGPI and Xanthan, respectively. Results showed the feasibility of preparation a low cholesterol mayonnaise by application a desirable combination of WGPI, XG, and egg, with comparable properties those of the conventional mayonnaise.