The application of a One Health approach recognizes that human health, animal health, plant health and ecosystem health are intrinsically connected. Tackling complex challenges associated with foodborne zoonoses, antimicrobial resistance, and emerging threats is imperative. Therefore, the One Health European Joint Programme was established within the European Union research programme Horizon 2020. The One Health European Joint Programme activities were based on the development and harmonization of a One Health science-based framework in the European Union (EU) and involved public health, animal health and food safety institutes from almost all EU Member States, the UK and Norway, thus strengthening the cooperation between public, medical and veterinary organizations in Europe. Activities including 24 joint research projects, 6 joint integrative projects and 17 PhD projects, and a multicountry simulation exercise facilitated harmonization of laboratory methods and surveillance, and improved tools for risk assessment. The provision of sustainable solutions is integral to a One Health approach. To ensure the legacy of the work of the One Health European Joint Programme, focus was on strategic communication and dissemination of the outputs and engagement of stakeholders at the national, European and international levels.