Background Agricultural biotechnology public-private partnerships (PPPs) have been recognized as having great potential in improving agricultural productivity and increasing food production in sub-Saharan Africa. However, there is much public skepticism about the use of GM (genetically modified) crops and suspicion about private sector involvement in agbiotech projects. This case study sought to understand the role of trust in the Bacillus thuringiensis (Bt) cotton in Burkina Faso project by exploring practices and challenges associated with trust-building, and determining what makes these practices effective from the perspective of multiple stakeholders. Methods We conducted semi-structured, face-to-face interviews to obtain stakeholders’ understanding of trust in general as well as in the context of agbiotech PPPs. Relevant documents and articles were analyzed to generate descriptions of how trust was operationalized in this evolving agbiotech PPP. Data was analyzed based on emergent themes to create a comprehensive narrative on how trust is understood and built among the partners and with the community. Results We derived four key lessons from our findings. First, strong collaboration between research, industry and farmers greatly contributes to both the success of, and fostering of trust in, the partnership. Second, this case study also revealed the important, though often unrecognized, role of researchers as players in the communication strategy of the project. Third, effective and comprehensive communication takes into account issues such as illiteracy and diversity. Fourth, follow-up at the field level and the need for a multifaceted communications strategy is important for helping push the project forward. Conclusions Burkina Faso’s well-established and effective cotton selling system laid the foundation for the implementation of the Bt cotton project – particularly, the strong dialogue and the receptivity to collaboration. Interviewees reported that establishing and maintaining trust among partners, researchers and the community in Burkina Faso greatly contributed to the success of the PPP. By addressing challenges to building trust and engaging in trust-building practices early on, improvements in the effectiveness of agbiotech PPPs are likely.