National movement behaviour guidelines, in isolation, are unlikely to influence practice or policy unless accompanied by robust knowledge mobilization (KMb) strategies. However, without pairing KMb strategies with systematic evaluation, the impact of large-scale dissemination is unknown. The objectives of this study were to (1) systematically assess the dissemination of the Canadian 24-Hour Movement Guidelines for Adults Aged 18-64 Years and Adults Aged 65 Years or Older (24HMG) using the Reach Effectiveness Adoption Implementation and Maintenance (RE-AIM) framework and (2) provide recommendations to support the KMb and future impact of national movement behaviour guidelines. Intermediary organizations involved in the development and dissemination of the 24HMG were invited to participate in this study. A combination of methods-including cross-sectional surveys, media monitoring, and website content analysis-were performed at multiple timepoints over a 12-month period to assess the dissemination of the 24HMG. Findings suggest that the multi-pronged dissemination approach used for the 24HMG had a large reach to guideline target audiences (approximately 11.9 million) but resulted in low awareness and knowledge of the 24HMG among adults living in Canada (31.9% and 1.6%, respectively). Dissemination activities performed by intermediary organizations peaked in the first 4-months post-guideline release (76% of responding organizations), trending downwards over time (53% of responding organizations at 12 months). The complexity of disseminating national movement behaviour guidelines presents many challenges to systemic adoption. However, the impact of future national movement behaviour guidelines may be improved by augmenting current dissemination approaches to include coordinated, scalable, and capacity-building strategies.