Leptospirosis is an endemic disease with moderate to high incidence in Mahasarakham province, Thailand. The present study was designed to assess the policy implementation mission regarding leptospirosis prevention and control from the national level to the local administrative levels, through a One Health perspective. A qualitative study was conducted, using documentation review, individual in-depth interviews with public health officers, local government officers, livestock officers who developed policy implementation tools or have responsibilities in leptospirosis prevention and control. The results show that Thailand has progressively developed a leptospirosis prevention and control policy framework at the national level, transferring the responsibility of its implementation to the local level. The province of Mahasarakham has decided to foster cooperation in leptospirosis prevention and control at the local level. However, there are insufficient linkages between provincial, district and sub-district departments to ensure comprehensive disease prevention activities at the local level concerning leptospirosis patients and the whole population.