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Abstract The world human population is more and more urban and cities have a strong impact on the biosphere. This explains the development of urban ecology. In this context, the goal of our work is fourfold: to describe the diversity of scientific questions in urban ecology, show how these questions are organized, to assess how these questions can be built in close interactions with stakeholders, to better understand the role urban ecology can play within ecological sciences. A workshop with scientists from all relevant fields (from ecology to sociology) and stakeholders was organized by the Foundation for Research on Biodiversity (FRB). Three types of scientific issues were outlined about (1) the biodiversity of organisms living in urban areas, (2) the functioning of urban organisms and ecosystems, (3) interactions between human societies and urban ecological systems. For all types of issues we outlined it was possible to distinguish both fundamental and applied scientific questions. This allowed building a unique research agenda encompassing all possible types of scientific issues in urban ecology. As all types of ecological and evolutionary questions can be asked in urban areas, urban ecology will likely be more and more influential in the development of ecology. Taken together, the future of towns, their biodiversity and the life of city dwellers is at stake. Increasing the space for ecosystems and biodiversity within towns is more and more viewed as crucial for the well-being of town dwellers. Depending on research and the way its results are taken into account, very different towns could emerge. Urban areas can be viewed as a test and a laboratory for the future of the interactions between human and ecological systems. Graphical abstract Unlabelled Image Highlights • A research agenda for urban ecology was built by scientists and stakeholders. • Questions on biodiversity, ecosystems, interactions with humans are distinguished. • For all types of issue it is possible to ask fundamental and applied questions. • Urban ecology will likely be more and more influential in the development of ecology. • The future of towns, their biodiversity and the life of city dwellers is at stake. [ABSTRACT FROM AUTHOR]