New helium isotopic data from hot springs on Shikoku Island were obtained from a larger number of sampling points than previous studies in order to elucidate the geographical distribution of 3He/4He ratios in the fore-arc regions of southwest Japan. Systematic, regional variations in 3He/4He ratios were observed on both Shikoku Island and the Kii Peninsula. The hot springs on Shikoku Island have 3He/4He ratios between 0.21 RA and 2.5 RA. On the other hand, the majority of hot springs in the Kii Peninsula have significantly higher 3He/4He ratios than those on Shikoku Island. It has been suggested that the anomalously high 3He/4He ratios in the fore-arc region of southwest Japan are related to the migration of MORB-type helium with aqueous fluids generated by dehydration of the subducting Philippine Sea plate and mixing with terrestrial waters (e.g., Matsumoto et al., 2003). According to recent seismological observations of crustal microearthquakes and their focal mechanisms, the geotectonic environment beneath Shikoku Island is subject to a stress field with no normal faulting and with extremely low crustal seismicity. This argues against migration of aqueous fluids from the subcrustal lithosphere, resulting in a relatively small influence of mantle-derived helium at the surface. As a consequence, the regional variation in 3He/4He ratios of hot springs on Shikoku Island is interpreted as a three-component mixture of mantle-derived helium associated with magmatism of middle Miocene of age (<3.4 RA), crustal, radiogenic helium and atmospheric helium.