Open innovation, understood as a strategy for business competitiveness, has experienced growing relevance, even in traditional economic sectors, such as agri-food. This article focuses on the trends and challenges of open innovation applied to the agri-food field, based on two approaches, a Systematic Literature Review (SLR) and a structured survey, answered mainly by micro-, small-, and medium-sized enterprises (MSMEs). For the SLR, the Scopus bibliographic database was chosen. Documents were filtered by type, novelty, and impact factor of the journal (based on the Scimago Rank). The final selection included 50 articles that were deeply analyzed. In addition, the survey was applied to 57 agri-food companies from the department of Nariño (Colombia), establishing a diagnosis of the extent of openness of their collaborative barriers and innovation capacities. The review’s results revealed a marked European dominance in this research field. Product co-creation, eco-innovation, and bioeconomy are main fields of interest and application of open innovation. The challenges identified are related to intellectual property rights and effective communication between stakeholders. The survey was successful in establishing a statistically significant correlation between innovation performance and collaboration with external partners. As a conclusion, an open innovation approach can provide dynamism and cohesion in agri-food systems.