Objective To investigate the dilemma and optimization path for implementation of the One Health concept at the grassroots level in China, so as to provide the decision-making evidence for grassroots health governance in China. Methods A total of 30 workers were sampled from grassroots government departments related to One Health in Beijing, Zhejiang, Hainan, and Qinghai from June to August in 2023, including provincial-, municipal-, and county-level health commissions, centers for disease control and prevention, departments of ecology and environment, agriculture departments, transportation departments, and administrations for market regulation) as interviewees. Semi-structured interviews were conducted using the convenience sampling method to collect practical experiences, current situations, and problems related to the multi-sectoral health collaboration of the One Health concept. The interview data were subjected to open coding, axial coding, selective coding and analysis using the software NVivo 12. Results Following open coding of interview data， a total of 51 initial concepts, along with 25 sub-categories were yielded, including preparation and response, safety risk, information exchange, concept acceptance, management and coordination, communication methods, social benefits, collaboration mechanisms, collaborative governance, cross-regional collaboration, policy and planning, incentive mechanisms, international cooperation, audit plans, departmental responsibilities, joint planning, cross-sectoral collaboration, information sharing, economic conditions, health conditions, publicity, resources, evaluation mechanisms, feedback mechanisms, and supervision and implementation. Following axial coding and re-clustering, these 25 sub-categories were condensed into 10 main categories, namely information exchange, policy and planning, incentive mechanisms, social benefits, dissemination, collaboration mechanisms, communication, resources, preparation and response, and international cooperation. Axial coding and selective coding revealed that multisectoral collaboration was notably crucial in implementation of the One Health concept, and this collaboration was embodied in cross-sectoral cooperation, emergency response, data sharing, social responsibility, and global collaboration. Conclusion Institutional innovation is critical to implementation of One Health, which requires the establishment of grassroots mechanisms to achieve health coordinated governance of “human-animal-environment”. It is necessary to implement the strategy of “preventing human diseases by controlling animal diseases, and implementation of interventions moving forward from the source”, and strengthen the prevention at the source. Future efforts should focus on expansion of pilot programs, popularization of assessment tools, and extension of policies to communities.