BackgroundThe implementation of the One Health (OH) approach improves the prevention and control of zoonotic diseases and ensures a better understanding of the emergence of infectious diseases. ObjectiveThis study aimed to identify the key stakeholders’ knowledge of the One Health concept and assess attitudes toward the need for collaboration and explore the viewpoints of stakeholders regarding One Health Collaboration. MethodsIt is an explanatory mixed-method study conducted in the Puducherry district, South India. The study involved quantitative data collection among 70 stakeholders from various departments using quota sampling. Followed by qualitative data exploration of the viewpoints of stakeholders on One Health Collaboration through key informant interviews among 10 key stakeholders selected using purposive sampling. Knowledge of the stakeholders on One Health was assessed by using a self-administered semi-structured questionnaire. Knowledge and attitudes were summarized as frequency and percentage. A manual content analysis of the transcripts of the interviews was done. ResultA total of 70 (Male-55.7 % & female-44.3 %) stakeholders were included in the study. 57.1 % of the stakeholders heard about One Health and the rest did not know. A few numbers of stakeholders (18.8 %) were aware of the formal connection between various departments. 86%of stakeholders suggested that intersectoral coordination is very much needed at the grass root level. The interviews found that a lack of standard guidelines, weak political commitment, poor departmental coordination, and a lack of community awareness were the most frequent challenges encountered. ConclusionThe operationalization of OH could be enhanced by providing standard guidelines and ensuring the coordination between the stakeholders.