Tritium is used as fuel for nuclear fusion reactions and has cancer risk for workers in nuclear fusion plant by its exposure during routine operation or accidents. Tritium exists as tritiated water (HTO) which is produced by an isotope exchange reaction from HT in normal circumstance. In this study we analyzed cellular response against HTO exposure in a human liver cancer cell line HepG2 and a cervical cancer cell line HeLa. These cells were immersed in RPMI medium containing HTO at the dose rate of 0.5 Gy/hr for 20 hrs (10 Gy). We determined whether the DNA-PK/AKT pathway is activated by chronic HTO exposure or not. As well as X-rays, exposure of HepG2 and HeLa cells to HTO activated AKT, which was shown by phosphorylated-AKT at Serine473. The activation of DNA-PK was also observed in these cells by X-ray irradiation but not by exposure to HTO. Instead of DNA-PK, epidermal growth factor receptor and ERK1/2 were activated following HTO exposure. These results suggested that certain cellular molecules are response to HTO exposure. Our study may provide molecular markers to estimate the biological effects in response to tritium exposure in human cells.