The objective of this study was to investigate the effect of pretreatment of canola meal with hydrothermal processing, phytase, and organic acid on phytate degradation. Six experiments were conducted processing canola meal in a hydrothermal reactor with different moisture conditions (15-350%), acid additions (hydrochloric acid, citric acid, malic acid, and lactic acid), pH (pH 4, 5, and 6), incubation times (30, 45, and 60 min), and phytase enzymes (‘Quantum Blue’ and ‘Finase’). The study revealed that although hydrothermal pretreatment of canola meal with phytase and higher moisture (200-350%) would allow 24.8-36% phytate breakdown, and a higher moisture addition (200%) combined with organic acid increased even further to 46.6%, lower moisture (50 and 100%) and organic acid was still effective in reducing phytate (by 7.9-19.4%). Optimal pH and incubation time in hydrothermal reactor for phytase efficacy were determined to be pH 4 and 5 and 30 to 60 min. Still, the results of the current study suggest that pretreatments should be further evaluated to optimize the efficacy of enzyme and organic acids as prebiotics to reduce anti-nutrients in canola meal, thus improving its utilization for livestock and reducing excretion to environment.