In response to a request from municipalities in the Lake Simcoe (Ontario) watershed, the Lake Simcoe Region Conservation Authority (LSRCA) has developed a toolkit on how to implement road ecology best management practices. The toolkit builds on previous work done by the LSRCA (e.g. mapping of potential vehicle-wildlife collision hotspots and testing of ecopassages), as well as recent studies and guidance completed in both local and global jurisdictions. The toolkit consists of template policies which can be easily adapted to fit within existing and future municipal planning tools (e.g. official plans, transportation master plans, etc.), as well as a guide to implementing ecopassages and a factsheet summarizing existing information. The policies were developed in collaboration with municipal, provincial and conservation authority partners from across Southern Ontario. These tools combine to assist project managers in implementing road ecology best management practices into the planning, design, construction and maintenance of road infrastructure projects. While the toolkit was designed for municipalities within Ontario, Canada, it can easily be adapted to fit the needs of other jurisdictions.