Ca2+/H+ antiporters play important roles in plant nutrition and signal transduction. A novel Ca2+/H+ antiporters gene OsCAX3 was identified from rice. Sequence analysis revealed that OsCAX3 has 11 transmembrane domains, and an acid motif consisting of 17 amino acids between sixth and seventh transmembrane domains. When expressed in yeast, OsCAX3 complemented the mutant growing deficiency at high Ca2+ concentrations, and its N-terminal(1~26 amino acid) partially suppressed its ability to transport Ca2+. RT-PCR analysis showed that OsCAX3 mRNA was induced by exogenous calcium. OsCAX3 was predicted to localize in plant plasma membrane by PSORT prediction, and this was further confirmed by transiently expressing OsCAX3-GFP fusion protein in mesophyll cell protoplast of Arabidopsis. All these findings strongly suggested that OsCAX3 is a Ca2+/H+ antiporter located in plasma membrane.