A profound transformation of agricultural production methods has become unavoidable due to the increase in the world’s population, and environmental and climatic challenges. Agroecology is now recognized as a challenging model for agricultural systems, promoting their diversification and adaptation to environmental and socio-economic contexts, with consequences for the entire agri-food system and the development of rural and urban areas. Through a prospective exercise performed at a large interdisciplinary institute, INRAE, a research agenda for agroecology was built that filled a gap through its ambition and interdisciplinarity. It concerned six topics. For genetics, there is a need to study genetic aspects of complex systems (e.g., mixtures of genotypes) and to develop breeding methods for them. For landscapes, challenges lie in effects of heterogeneity at multiple scales, in multifunctionality and in the design of agroecological landscapes. Agricultural equipment and digital technologies show high potential for monitoring dynamics of agroecosystems. For modeling, challenges include approaches to complexity, consideration of spatial and temporal dimensions and representation of the cascade from cropping practices to ecosystem services. The agroecological transition of farms calls for modeling and observational approaches as well as for creating new design methods. Integration of agroecology into food systems raises the issues of product specificity, consumer behavior and organization of markets, standards and public policies. In addition, transversal priorities were identified: (i) generating sets of biological data, through research and participatory mechanisms, that are appropriate for designing agroecological systems and (ii) collecting and using coherent sets of data to enable assessment of vulnerability, resilience and risk in order to evaluate the performance of agroecological systems and to contribute to scaling up. The main lessons learned from this collective exercise can be useful for the entire scientific community engaged in research into agroecology.