Shifting dietary patterns from animal-based proteins to more plant-based and alternative protein sources – the protein transition – is urgently needed to improve planetary and human health. Public food procurement is considered to be an effective policy instrument to accelerate the protein transition and to be a potential game changer towards a sustainable food system. However, this potential has remained far from leveraged, and it is largely unknown which barriers and enablers exist in that context. Therefore, this study aimed to gain insight into the barriers and facilitators that are perceived by relevant stakeholders when implementing the protein transition in public food procurement. Our study was conducted in the Netherlands, because of the policy goals set by the Dutch government with regard to the protein transition (50% of the proteins consumed should include animal-based proteins and 50% plant-based proteins by 2030) and because the extent to which the protein transition has been included in the food procurement of Dutch (semi-)public organizations is still largely unknown. However, findings are also relevant for other countries. Semi-structured interviews were conducted with participants from a wide range of (semi-)public organizations (e.g., universities, hospitals, governments), in 2022. Five main themes of barriers and facilitators were identified: (1) support and motivation, (2) food availability and offerings, (3) financial considerations and incentives, (4) policies, processes, and contracts, and (5) environmental factors. The insights from this study can strengthen the scientific evidence base and can serve as a foundation for future research. Moreover, the insights can be beneficial to officials working in (semi-)public organizations to effectively design and execute their procurement process, and can help policymakers in policy development to foster (semi-)public organizations to implement the protein transition in their own contexts.