This paper provides an in-depth review of experiences and insights from mainstreaming biodiversity and development in South Africa. More specifically, it describes how biodiversity considerations have been mainstreamed in five key sectors/areas, namely: land use planning, mining, water, infrastructure, and the agricultural sector. It discusses the types of barriers and challenges that have been encountered, the key ingredients and lessons learned to help ensure more effective biodiversity mainstreaming, and the role of development co-operation in supporting in mainstreaming in South Africa. Examples of the key elements of success include good science, the ability to harness windows of opportunity, and ensuring genuine links to development objectives.