We studied changes in water chemistry along the groundwater flow path in the Matsumoto tunnel vicinity and estimated the origin of the groundwater and dissolued substances. The relationship between the concentration of HCO3-, Ca2+, and Na+, and the CO2 gas pressure in the groundwater indicated that the HCO3-, Ca2+, and Na+ were produced by reaction of the CO2 gas in the groundwater and feldspar in the rocks. The relationship between the concentration of NO3- and the Eh and pH values in the groundwater indicated that in an oxidative condition, some bacteria used NH4+ and produced NO3- and H+ and in a reductive condition, other bacteria used NO3- and produced N2 gas and OH-. The stable hydrogen and oxygen isotopic ratios in the groundwater and precipitation indicated that the groundwater originated from precipitation fell on the area. The concentration of 3H and the stable hydrogen and oxygen isotopic ratios in the groundwater suggested that it was getting warmer for more than 60 years. The stable carbon isotopic ratio indicated that the HCO3- in the groundwater originated from CO2 gas produced by organic matter in the soil except in the deep well water. The deep well water, which had a higher concentration of HCO3- than the other groundwater sampled was thought to have acquired HCO3- through contact with rocks. The stable sulfur isotopic ratio indicated that the SO42- in the groundwater was of the same origin.