Amebiasis is a notifiable infectious disease in Japan, and the number of reported cases had been on the rise, but since the discontinuation of insurance-covered serum antibody testing reagent in 2017, concerns have arisen regarding the decrease in reported cases. This study aimed to investigate changes occurring after discontinuation of the serum antibody test reagent production. We retrospectively analyzed amebiasis cases from January 2014 to December 2019 using the National Center for Epidemiology of Infectious Diseases system. Interrupted time-series regression analysis was used to evaluate trends in weekly amebiasis cases before and after the discontinuation period. The study period was divided into prediscontinuation (2014-2017) and discontinuation (2018-2019) periods. A total of 6,179 amebiasis cases were reported. The average numbers of weekly cases were 21.5 during 2014-2017 and 16.3 during 2018-2019. The frequency of diagnoses decreased in the discontinuation period (prevalence rate ratio = 0.78; 95% CI, 0.67-0.89; P < 0.01). Subgroup analysis showed lower diagnostic rates, particularly for extraintestinal amebiasis (prevalence rate ratio = 0.37; 95% CI, 0.22-0.55; P < 0.01). We observed a significant decrease in the number of reported amebiasis cases per week after discontinuation of the serum antibody test reagent in Japan. Our findings hold significance for both public health policy and practice in Japan, underscoring the requirement for enhanced amebiasis diagnostic tools and strategies. To ensure accurate diagnosis, availability of antibody reagents for serum testing, covered by insurance, should be encouraged.