Canola meal (CM) contains a significant amount of non-starch polysaccharides (NSP) which with the use of enzyme technology could be hydrolyzed to low-molecular-weight polysaccharides and oligosaccharides. We hypothesized that enzymatically-released bioactive components of CM fibre would have a prebiotic effect on gastrointestinal microbiota of monogastric animals. A variety of carbohydrases were evaluated in the series of in vitro studies, and the most effective enzyme combination was used in the study. The blend of a multi-carbohydrase preparation containing pectinase and xylanase activities showed the ability to hydrolyze 47.9% of CM NSP in vitro and improved NSP digestibility of broilers fed diets containing CM. The enzyme blend was used to produce enzymatically-modified CM (ECM, lab scale) and ECM solubles (ECMS, pilot scale), and their prebiotic properties were evaluated. The results of the broiler chicken study showed that replacing CM with ECM significantly improved the overall apparent total tract digestibility of NSP. Lactobacillus spp. was present in much larger counts (P < 0.01) in ileal and cecal digesta of birds fed the ECM diets when compared to the Control treatment. The AMEn value of ECM was 31% higher than that of CM. There was no difference in the standardized ileal amino acid digestibility of CM and ECM. Supplementation diets with ECMS improved growth performance, increased total SCFA in ileal digesta, and provided favorable conditions for the proliferation of Lactobacillus spp. and Enterococcus spp. in ileum and ceca of broilers. The effect of ECM and ECMS supplementation on growth performance and gut health of weaned piglets was investigated. No effect of ECM was observed; however, ECMS positively affected the intestinal pH and proliferation of beneficial gut microbiota. In conclusion, modification of CM fibre with carbohydrases significantly changed its structure and enhanced its bioactivity. Processing of CM developed throughout this project generated novel products, ECM and ECMS, which when fed to broilers and pigs, demonstrated their ability to enhance the diversity of the gut microbiota population and improve nutrient utilization. Therefore, the enzymatic modification of CM can create value-added feed additives, which can improve animal health and support antibiotic-free feeding programs in monogastric animals.