The St. Marys River is a 112 km connecting channel between Lake Superior and Lake Huron. The river and its associated marshes, riparian areas, and upland habitats, comprise one of the most biologically diverse regions in the Great Lakes Basin, and the area was identified as a priority for conservation by The Nature Conservancy (TNC) in 2000. Using TNC’s Conservation Action Planning (CAP) process, we identified important conservation targets, developed practical indicators to track their current and future status, identified threats to target species and ecosystems, explored opportunities for the conservation of the targets given the social, economic, political and cultural environment, and developed strategies to protect, enhance, or restore biodiversity. We used a variety of research methods including extensive literature reviews, interviews, and focus groups with conservation professionals in the St. Marys River basin to incorporate their local and regional expertise into our project. The outcome of this project is an adaptive management plan to inform TNC’s future involvement in biodiversity conservation in the St. Marys River region.