The relationship between geological structure and helium isotopes is discussed for deep groundwaters from the Osaka sedimentary basin, southwest Japan, to understand dissolved He in groundwater for hydrological application. Although this area shows no Quaternary volcanic activity, nearly upper mantle-like ³He/⁴He ratio (1.1 × 10^-5) has been observed in the area along the fault system where the basement rocks are outcropped. In contrast, deep groundwaters from the lowest part of the aquifers beneath the Osaka Basin showed a wide variation in ³He/⁴He ratio (0.27-8 × 10^-6), which seems to reflect the geological structure. The moderately high ³He/⁴He ratios, not as high as those in the mantle value, are due to a contribution of radiogenic ⁴He within the aquifer. The flux of mantle He through the Uemachi thrust fault into the aquifers in the east block (hanging wall of the fault) of the Osaka Basin will be smaller than those in the west block (foot wall), because mantle He through this fault encountered the aquifers in the west block and dissolved in this aquifer. The model for the Osaka Basin presented in this study implies that there are at least two different sources of He flux into an aquifer; mantle He flux through the fault system and crustal He from the underlying formation. The former should be spatially heterogeneous, while crustal ⁴He flux is rather spatially constant throughout the lowermost part of the Osaka Basin.