We used high-performance liquid chromatography to separate urine obtained from whole-body gamma-irradiated mice (4 Gy) before analyzing each fraction with matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry to identify radiation-responsive molecules. We identified two candidates: hepcidin antimicrobial peptide 2 (hepcidin-2) and peptide fragments of kidney androgen-regulated protein (KAP). We observed that peak increases of hepcidin-2 in urine were delayed in a dose-dependent manner (1 Gy and above); however, the amount of KAP peptide fragments showed no correlation with radiation dose. In addition, an increase in hepcidin-2 after exposure to relatively low radiation doses (0.25 and 0.5 Gy, respectively) was biphasic (at 8-48 h and 120-168 h, respectively, after irradiation). The increase in hepcidin-2 paralleled an increase in hepcidin-2 gene (Hamp2) mRNA levels in the liver. These results suggest that radiation exposure directly or indirectly induces urinary excretion of hepcidin-2 at least in part by the upregulation of Hamp2 mRNA in the liver.