In previous study, a cDNA library enriched for mRNAs encoding ESTs that increased in abundance during infection with Phytophthora sojae was constructed by suppression subtractive hybridization from leaf tissues of a high resistant soybean, and an EST homologous to the class 10 of pathogenesis-related (PR) proteins was identified to be up-regulated by microarray and real-time PCR. Here, the full-length cDNA (termed GmPR10, GenBank accession number FJ960440; ADC31789.1) of the EST was isolated by rapid amplification of cDNA ends, and contains an open reading frame of 474 bp. The GmPR10 protein included a "P-loop'' motif. The constitutive transcript abundance of GmPR10 in soybean was the highest in leaves, followed by roots and stems. Further analysis showed that GmPR10 mRNA abundance was increased during infection with P. sojae following leaf treatments with gibberellin (GA3), hydrogen peroxide (H2O2), salicylic acid (SA), and abscisic acid (ABA). The dialytically renatured GmPR10 protein significantly inhibited P. sojae hyphal growth and exhibited RNase activity. Transgenic tobacco and soybean plants overexpressing GmPR10 showed increased resistance to P. nicotianae Breda and P. sojae, respectively. These results suggest that the GmPR10 protein plays an important role in host defense against P. sojae infection. To the best of our knowledge, this is the first report on the functional characterization of a PR10 protein from soybean in defense against P. sojae.