Leptospirosis is a zoonotic disease that the pathogen can be transmitted to humans through the excretions of infected animals. In the Republic of Korea, the Livestock Manure Control Act was enforced in September 2007 to improve underground water hygiene. The objective of this study was to evaluate the impact of Livestock Manure Control Policy on the incidence and the trend of human leptospirosis. An interrupted time series analysis using the monthly incidence of leptospirosis was conducted based on data derived from the Korean National Surveillance System between January 1999 and January 2015. We used a Spearman correlation method to compare the level of leptospirosis incidence decrease between the metropolitan cities and rural provinces. The annual incidence of leptospirosis in South Korea decreased by 33% after policy enforcement of the policy. A significant change in the slope of human leptospirosis cases was observed after the policy enforcement (β = −0·09, P < 0·001). Moreover, we detected a clear association between the size of the rice paddy fields and the decrease in leptospirosis incidence in provinces (r = 0·817, P = 0·01). This study shows that the Livestock Manure Control Policy had significantly reduced human leptospirosis incidence in the Republic of Korea, in particular, in rural regions.