One Health approach recognizes the intricate relationship between the health of man, animals, and their shared environment. It integrates efforts of multiple disciplines working locally, nationally, and globally to achieve optimal health for people, animals, and the environment. The changing interactions between people, animals, plants, and the environment have pushed the growth and expansion of human populations into new geographic areas, forcing people to live close to wild and domestic animals. The proximity, climate change, and land use have led to a disrupted environment that provides opportunities for zoonoses. About 60% of all human infectious diseases and approximately 75% of emerging infections are zoonoses. These diseases cause endemic health burdens in low-income settings, pressure on healthcare infrastructure, community tensions, and decreased economic prosperity. Control of zoonoses requires the multisectoral collaboration of experts in human, animal, and ecosystem services under a One Health approach. Information from this study can inform one health policy revision leading to the rational use of scarce resources and achieving health equity. However, such benefits cannot be achieved without a functional county-level One Health unit, as in Kisumu. A cross-sectional study generated information from 142 respondents regarding factors influencing multisectoral collaboration under a One Health approach in controlling zoonoses in Kisumu County. The nature, extent, and barriers to collaboration, captured on a semi- structured self-administered questionnaire, were determined and assessed using descriptive and inferential statistics. Multisectoral collaboration to control zoonoses in the area was average (51.4%) and driven mainly by public health officers. Conducting joint sensitizations [x2 (5) = 25.329, p <0.001)], notifying other sectors whenever a zoonosis is encountered [x2 (5) = 44.265, p <0.000)] and collaboration through professional associations [x2(5) = 17.776, p <0.001)] were the significant nature of collaboration. Lack of communication [H (5) =23.055, p < 0.001)], Lack of transport facilities [H (5) = 23.588, p < 0.001)], lack of interest in zoonoses at work and during training [H (5) = 25.678, p <0.001)], insufficient funding [H (5) = 34.453, p = 0.001)], differences in emphasis on zoonoses at the across the sectors [H (5) = 21.068, p < 0.001)] were the significant factors affecting One Health collaboration. Respondents observed that communication among OHCs is can enhance cooperation, transport facilities are needed to enhance responses where collaboration is required, funding is needed to support one health operations and continued specialization increases focus area, thus hindering collaboration. Addressing staffing requirements, training needs, allocation of resources, and setting up a robust information management system for data sharing can improve multisectoral collaboration for the One Health approach