The innovation literature is growing rapidly, and a primary concern revolves around the contexts that foster and enhance innovation and entrepreneurial outcomes. Research on innovation environments still requires further development. This paper sheds light on fostering and developing innovation and entrepreneurial ecosystems in cities from the institutional work perspective, emphasizing the need to institutionalize collaboration. The paper employs the case study method with a process data approach. The study took place in two Latin American cities, Porto Alegre and Florianópolis, within a cultural context characterized by a non-cooperative background. The findings are linked to the institutionalization of collaboration in the development of innovation and entrepreneurial ecosystems, unfolding in three phases involving three groups of roles: leaders, supporters, and the community. The practices are associated with the leader's group in the first phase (Preparing for Collaboration). The second phase (Strengthening Collaboration) expands the practices and adjusts the focus to the supporter's group. Additionally, the third phase (Disseminating Collaboration) continues to broaden the scope and gains relevance for the community's group. Therefore, the evolution of the phases is connected to the expansion of the range of institutionalization practices and a focus on the most active roles. These results carry significant implications for the theory and practice of innovation and entrepreneurial ecosystem emergence, offering insights into the phases and roles critical for fostering innovation environments. Furthermore, they can provide practical guidance for other cities or regions aiming to cultivate robust innovation and entrepreneurial ecosystems. • The process of fostering and developing an innovation an entrepreneurial ecosystem in cities with the institutional work perspective considers the need to institutionalize the collaboration. • The study was conducted in two cities with a cultural context of non-cooperation background. • Institutionalizing collaboration for the innovation and entrepreneurial ecosystem occurs in three phases: Preparing for Collaboration, Strengthening Collaboration and Disseminating Collaboration. • Innovation and entrepreneurial ecosystem development involve three groups of roles: leaders, supporters, and community. • The evolution of the phases is related to the expansion of the range of institutionalization practices and to the focus on the roles most involved. [ABSTRACT FROM AUTHOR]