Understanding the origin of acid mine drainage (AMD) in a closed mine and groundwater flow system around the mine aids in developing strategies for environmental protection and management. AMD has been continuously collected and neutralized at Osarizawa Mine, Akita Prefecture, Japan, since the mine was closed in the 1970s, to protect surrounding river water and groundwater quality. Thus, water samples were taken at the mine and surrounding groundwaters and rivers to characterize the chemical properties and environmental isotopes (δ²H and δ¹⁸O). The results showed that the quality and stable isotope ratios of AMD differed from those of groundwater/river water, indicating that the recharge areas of AMD. The recharge area of AMD was evaluated as the mountain slope at an elevation of 400–500 m while that of the surrounding groundwater was evaluated at an elevation of 350–450 m, by considering the stable isotopes ratios. This indicates that the groundwater affected by AMD is limited to the vicinity of the mine and distributed around nearby rivers.