Quasi-periodic sampling and analysis of spring water were conducted at nine sites to investigate the effects of the 2016 Kumamoto earthquake on the major chemical composition, including hydrogen, and oxygen stable isotopic ratios, of spring waters. Transient increases in the concentrations of nitrate in spring waters were observed at seven sites in May 2016, one month after the earthquake. This was the first time that this phenomenon had been observed on such a large scale. The magnitude of the increase in nitrate concentration was 0.7-2.4 mg/L, equivalent to 10%-93% of the concentration before the earthquake. Furthermore, precipitation data showed that heavy rain of over 70 mm/day occurred 5 days after the mainshock. Rainwater is thought to have transported nitrate from shallow underground soil to the groundwater aquifer through cracks caused by strong shaking during the earthquake. Strong shaking also caused damage to the plants, decreasing the amount of nitrate they absorbed, and this is thought to be one of the reasons for the increase in nitrate concentration in the springs.