Background The Bacillus thuringiensis (Bt) cotton public-private partnership (PPP) project in East Africa was designed to gather baseline data on the effect of Bt cotton on biodiversity and the possibility of gene flow to wild cotton varieties. The results of the project are intended to be useful for Kenya, Uganda, and Tanzania when applying for biosafety approvals. Using the backdrop of the different biosafety regulations in the three countries, we investigate the role of trust in the Bt cotton partnership in East Africa. Methods Data were collected by reviewing relevant project documents and peer-reviewed articles on Bt cotton in Tanzania, Kenya and Uganda; conducting face-to-face interviews with key informants of the project; and conducting direct observations of the project. Data were analyzed based on recurring and emergent themes to create a comprehensive narrative on how trust is understood and built among the partners and with the community. Results We identified three factors that posed challenges to building trust in the Bt cotton project in East Africa: different regulatory regimes among the three countries; structural and management differences among the three partner institutions; and poor public awareness of GM crops and negative perceptions of the private sector. The structural and management differences were said to be addressed through joint planning, harmonization of research protocols, and management practices, while poor public awareness of GM crops and negative perceptions of the private sector were said to be addressed through open communication, sharing of resources, direct stakeholder engagement and awareness creation. The regulatory differences remained outside the scope of the project. Conclusions To improve the effectiveness of agbiotech PPPs, there is first a need for a regulatory regime that is acceptable to both the public and private sector partners. Second, early and continuous joint planning; sharing of information; and transparency encourages accountability and fosters trust building. Third, direct stakeholder engagement and awareness creation builds trust between project partners and the community. A concern raised by the interviewees was the absence of a regulatory framework in Tanzania, which deterred active private sector participation in the project.