Public health systems have critical and clear relevance to the World Bank’s twin goals of poverty eradication and boosting shared prosperity. In particular, they are impacted by, and must respond to,significant threats at human-animal-environment interface. Most obvious are the diseases shared between humans and animals (“zoonotic” diseases), which comprise more than 60 percent of known human infectious pathogens; but also aspects of vector-borne disease, food and water safety and security, and antimicrobial resistance. Public health systems must therefore be resilient and prepared to face existing and future disease threats at the human-animal-environment interface. the Operational Framework provides a strong orientation to One Health to assist users inunderstanding and implementing it, from rationale to concrete guidance for its application. Six core chapters are included, supported by annexes diving deeper into operational tools and recent World Bank alignment with One Health topics, and a glossary that explains key terms, including interpretations specific to the Operational Framework. Chapter one presents background on the need and scope for One Health, showing how it is inclusive of and can be useful in addressing a broad range of priorities for human and animal health and environment sectors. Chapter two reviews the economic argument for One Health for the global and local public good – both through more effective disease prevention and control, as well as operational efficiencies at countryand project level. Chapter three showcases relevant tools and initiatives for One Health that support capacity for human, animal and or environmental health sectors, bringing them together and articulating possible connections as well as identifying priority areas for further development to aid in successful One Health operations, with additional examples provided in the Annex. Chapters four to six present specific applications of One Health. Examples of entry points for One Healththinking are shown in Chapter four, including determining relevance of different sectors for involvement based on the specific context. Chapter five outlines the building blocks for embedding One Health approaches to prepare for endemic, emerging and pandemic threats, all the way from disease prevention to recovery. Finally, noting the challenge of monitoring progress across sectors, Chapter six outlines possible pathways for monitoring and upscaling, showcasing indicators from relevant Bank projects. The Operational Framework is intended as a guide for One Health operations, from project and program scoping and identification stages to design and implementation, including monitoring and evaluation, to help optimize investments.