Abstract In addition to establishing methods for proteome analysis of cell wall proteins (CWPs) for the model plant Medicago truncatula, this work highlights the presence of several protein classes in cell culture. Using a combination of two-dimensional gel electrophoresis (2D-PAGE) and/or liquid chromatography–tandem mass spectrometry (LC–MS/MS), we established the proteome reference map of M. truncatula cell wall proteins. CWPs extracted from purified cell wall fragments resulted in the identification of 46 (2D-PAGE) and 65 (LC–MS/MS) proteins, respectively, with a total of 111 proteins. The identified proteins are involved in various processes, including cell wall modifications, signaling, defense mechanisms, membrane transport, protein synthesis and processing. Further, we conducted comparative proteome analysis to identify changes in protein composition during interaction of M. truncatula cell suspension culture with a pathogen-derived yeast elicitor (YE) and suppressor using Sinorhizobium meliloti LPS. 2D-PAGE analysis for the CWPs after YE and LPS treatment resembled the proteome map of YE alone, with a few up-regulated proteins involved in defense, and in the case of the LPS-treated cell wall proteome, there was no significant difference observed. Using this approach, proteins involved in defense, such as l-ascorbate peroxidase, specifically targeted proteins to the cell wall during defense, including glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and proteins that play an important role during growth and development were identified. Also, some defense-related proteins were absent in the same gel after YE treatment, suggesting that oxidant protection is regulated by these proteins.