Mesophotic coral ecosystems (MCEs) and temperate mesophotic ecosystems (TMEs) have received increasing research attention during the last decade as many new and improved methods and technologies have become more accessible to explore deeper parts of the ocean. However, large voids in knowledge remain in our scientific understanding, limiting our ability to make scientifically based decisions for conservation and management of these ecosystems. Here, we present a list of key research and conservation questions to enhance progress in the field. Questions were generated following an initial open call to MCE and TME experts, representing a range of career levels, interests, organizations (including academia, governmental, and nongovernmental), and geographic locations. Questions were refined and grouped into eight broad themes: (1) Distribution, (2) Environmental and Physical Processes, (3) Biodiversity and Community Structure, (4) Ecological Processes, (5) Connectivity, (6) Physiology, (7) Threats, and (8) Management and Policy. Questions were ranked within themes, and a workshop was used to discuss, refine, and finalize a list of 25 key questions. The 25 questions are presented as a guide for MCE and TME researchers, managers, and funders for future work and collaborations.