The ultrafiltration/diafiltration (UF/DF) process is a crucial step in the canola protein isolation process from rapeseed meal. The process involves using a multi-stage membrane system to separate components of the mixture. As diafiltration dilutes the feed stream in the ultrafiltration system, a large amount of diafiltration water is required. Reducing the diafiltrate for sustainability reasons can be done by carefully selecting process variables or using recycle streams. However, finding the optimum process variables can be a meticulous process if performed experimentally or via trial and error. In this study, we performed an optimization using a mechanistic model integrated with a genetic algorithm to aid in finding an optimum combination of process variables. The mechanistic ultrafiltration model was derived by taking into account transport phenomena within the filtration system. Parameters were characterized experimentally in term of viscosity coefficient, membrane resistance, cake porosity, aggregate diameter equivalent, and material compressibility factors. Using the mechanistic model-based optimization in combination with actual experimental values, the performance of a four-stage UF/DF system could already be improved despite fixing the configuration, albeit at the cost of a reduction in purity. Further improvement could be achieved by using recycle streams. The optimized system achieved a diafiltrate reduction of up to 79%, an increase of purity of up to 31%, and an increase of dry matter content of up to 18%, while maintaining the product purity of the reference set-up.