This study aimed to identify the proteins produced specifically in Betula platyphylla var. japonica plantlet No. 8 by infection with Inonotus obliquus strain IO-U1. Sterile Japanese birch plantlets were infected with the fungus, and the protein samples obtained at 2 days post-infection were subjected to two-dimensional electrophoresis to detect infection-specific proteins. The specific proteins were analyzed using MALDI-TOF-MS and identified by peptide mass fingerprinting with MASCOT software. Among the 735 protein spots detected in the infected plantlets, 169 spots were recognized as infection-specific proteins. Of these spots, 91 were analyzed using MALDI-TOF-MS, with two heat shock proteins (Hsp70 and Hsp60) being identified as infection-specific proteins. Hsp70 and Hsp60 may cooperate to refold proteins that were denatured by the infection of I. obliquus strain IO-U1 in the birch plantlets. It is suggested that these proteins are expressed in B. platyphylla plantlet No. 8 by infectious stress with I. obliquus strain IO-U1.