Regulatory requirements for renewable content in diesel fuel have been adopted in Canada. Fatty acid alkyl esters, that is, biodiesel, will likely be used to meet the regulations. However, the impacts on ambient atmospheric pollutant concentrations and human health outcomes associated with the use of biodiesel fuel blends in heavy duty diesel vehicles across Canada have not been evaluated. The objective of this study was to assess the potential human health implications of the widespread use of biodiesel in Canada compared to those from ultralow sulfur diesel (ULSD). The health impacts/benefits resulting from biodiesel use were determined with the Air Quality Benefits Assessment Tool, based on output from the AURAMS air quality modeling system and the MOBILE6.2C on-road vehicle emissions model. Scenarios included runs for ULSD and biodiesel blends with 5 and 20% of biodiesel by volume, and compared their use in 2006 and 2020. Although modeling and data limitations exist, the results of this study suggested that the use of biodiesel fuel blends compared to ULSD was expected to result in very minimal changes in air quality and health benefits/costs across Canada, and these were likely to diminish over time.