AimsThe objective of this study was to determine the optimal pretreatment and fungal strain to reduce glucosinolates (GLS), fibre and residual sugars while increasing the nutritional value of canola meal. Methods and ResultsSubmerged incubation conditions were used to evaluate four pretreatment methods (extrusion, hot water cook, dilute acid and dilute alkali) and three fungal cultures (Aureobasidium pullulans Y-2311-1, Fusarium venenatum NRRL-26139 and Trichoderma reesei NRRL-3653) in hexane-extracted (HE) and cold-pressed (CP) canola meal. ConclusionsThe combination of extrusion pretreatment followed by incubation with T. reesei resulted in the greatest overall improvement to HE canola meal, increasing protein to 515%, while reducing NDF, GLS and residual sugars to 186%, 172moll(-1)g(-1) and 5% w/w, respectively. Extrusion pretreatment and incubation with F. venenatum performed the best with CP canola meal, resulting in 544% protein while reducing NDF, GLS and residual sugars to 116%, 67moll(-1)g(-1) and 38% w/w respectively. Significance and Impact of the StudyThe work is significant in that it provides a method of reducing GLS (up to 98%) and neutral detergent fibre (up to 65%) while increasing the protein content (up to 45%) of canola meal. This novel pretreatment and submerged incubation process could be used to produce a canola product with higher nutritional value for livestock consumption.