This article explores attitudes of stakeholders involved in biotechnology towards the Responsible Innovation (RI) framework. As a framework for governance, RI has received increasing scholarly attention but has yet to be successfully integrated into U.S. research and innovation policy. Using a mixed methods approach, we analyzed the attitudes of different biotechnology stakeholders, particularly those working in areas related to genetically modified organisms (GMOs) in agriculture and the environment, towards the principles and practices of RI. Homogenous focus groups (organized by stakeholder affiliation) and pre-and post-focus group surveys were used to measure attitudes towards RI. We designed the survey questions according to the Advocacy Coalition Framework (ACF) and examined the agreement of stakeholders with policy core beliefs (general principles of RI) and secondary beliefs (implementation practices of RI). Although all stakeholder groups had neutral to positive attitudes towards RI general principles, we found significant differences in their reactions to the scholarly definitions of RI and in their attitudes towards practices to implement RI. In comparison to government and advocacy groups, stakeholders promoting biotechnology innovations-industry, trade organizations, and academics-had more negative reactions to social science definitions of RI and to RI practices that relinquish control to people outside of technology development pipelines. Qualitative analysis of focus-groups revealed barriers for implementing RI practices. For example, innovators were cynical about including external voices in innovation pathways due to inflexible funding programs and were concerned about potential delays to innovation given the highly competitive environments for financing and patents. In order to help address these tensions, we call for the co-design of RI practices between biotechnology innovators and other stakeholders. The opening-up of biotechnology innovation to RI practices of anticipation, inclusion, responsiveness and reflexivity will likely be important for future, public legitimacy of emerging genetic engineering applications such as gene editing and gene drives.