We investigated the effects of carbon ion and gamma-irradiation on osteoblastic MC3T3-E1 cells by comparing mRNA expression levels for RANKL and osteoprotegerin by RT-PCR. MC3T3-E1 cells were irradiated with 2, 4, or 6 Gy of carbon ions or gamma-rays, and total RNA was harvested 1, 2, 3, 5, or 7 days after irradiation. The RANKL mRNA/OPG mRNA ratio in carbon ion-irradiated MC3T3-E1 cells was lower, while in gamma-irradiated MC3T3-E1 cells this ratio was higher than in non-irradiated cells. To evaluate osteoclastogenesis of MC3T3-E1 cells, carbon ion- or gamma-irradiated cells were co-cultured with non-irradiated cells from murine bone marrow. Staining for tartrate-resistant acid phosphatase (TRAP) in co-cultures showed that carbon ion irradiation suppressed osteoclastogenesis. This result is consistent with the lower RANKL/OPG mRNA ratio for carbon ion-irradiated cells. These results suggest that carbon ion irradiation acts primarily on osteoblastic cells, leading to a decrease in the RANKL/OPG mRNA ratio. This effect, in turn, leads to a decrease in osteoclastogenesis and osteoclast activity, which results in an increase in bone volume.