Black spot disease in poplar is a disease of the leaf caused by fungus. The major pathogen is Marssonina brunnea f. sp. multigermtubi. To date, little is known about the molecular mechanism of poplar (M. brunnea) interaction. In order to identify the proteins related to disease resistance and understand its molecular basis, the clone “NL895” (P. euramericana CL“NL895”), which is highly resistant to M. brunnea f. sp. multigermtubi, was used in this study. We used two-dimensional gel electrophoresis (2-DE) and mass spectrometry (MS) to identify the proteins in poplar leaves that were differentially expressed in response to black spot disease pathogen, M. brunnea f. sp. mul- tigermtubi. Proteins extracted from poplar leaves at 0, 12, 24, 48, and 72 h after pathogen-inoculation were separated by 2-DE. About 500 reproducible protein spots were detected, of which 40 protein spots displayed differential expression in levels and were subjected to Ma- trix assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) followed by database searching. According to the function, the identified proteins were sorted into five categories, that is, protein synthesis, metabolism, defense response and un- classified proteins. Black spot disease in poplar is a disease of the leaf caused by fungus. The major pathogen is Marssonina brunnea f. Sp. Multigermtubi. To date, little is known about the molecular mechanism of poplar (M. brunnea) interaction. In order to identify the proteins related to disease resistance and understand its molecular basis, the clone “NL895” (P. euramericana CL “NL895”), which is highly resistant to M. brunnea f. sp. multigermtubi, was used in this study . We used two-dimensional gel electrophoresis (2-DE) and mass spectrometry (MS) to identify the proteins in poplar leaves that were differentially expressed in response to black spot disease pathogen, M. brunnea f. Sp. Mul-tiger mtubi. Proteins About 500 reproducible protein spots were detected, of which 40 protein spots showed differential expression in levels and were subjected to Ma - trix assisted laser desorption / ionizati on time of flight mass spectrometry (MALDI-TOF MS) followed by database searching. According to the function, the identified proteins were sorted into five categories, that is, protein synthesis, metabolism, defense response and un- classified proteins.