One Health is a concept which fosters collaborative relationships between human health, animal health and environmental health partners. Diseases are emerging and re-emerging in South Asia due to poor sanitation, close proximity of people to livestock, deforestation, porous borders, climate change, changes in human behaviour and unhygienic food preparation and consumption practices. This review was completed in two stages. First, we conducted a review of peer-reviewed literature and grey literature available in Google search engine related to One Health in four countries (Bangladesh, Bhutan, India and Nepal). Second, we used a structured questionnaire completed by the key stakeholders working on One Health for the collection of information related to the challenges in implementing One Health. Most of the One Health activities in South Asia are determined by donor preferences. Bangladesh and India did considerable work in advancing One Health with limited support from the government agencies. Weak surveillance mechanisms, uncertain cost-effectiveness of One Health compared with the existing approach, human resources and laboratory capacity are some of the factors hindering implementation of the One Health concept. Implementation of One Health is growing in the South Asia region with limited or no government acceptance. To institutionalise it, there is a need for leadership, government support and funding.