The current study aimed to detect and identify significant differentially expressed proteins between a virulent and an attenuated Histomonas meleagridis strain which was in vitro co-cultivated with Escherichia coli DH5α. Two-dimensional gel electrophoresis (2-DE) was used for proteome visualization , gel image software for computational detection of significantly up-regulated protein spots and matrix-assisted laser desorption/ionization time-of-flight/time-of-flight mass spectrometry (MALDI-TOF/TOF MS) for protein identification. The statistical analysis fulfilling two criteria (> or = 3-fold up-regulation and P<0.05) detected 119 differentially expressed protein spots out of which 62 spots were located in gels of the virulent strain and 57 spots in gels of the attenuated strain. The mass spectrometric analysis of 32 spots, up-regulated in gels of the virulent strain, showed that they are of H. meleagridis origin. As opposed to this, the mass spectrometric analysis of 49 protein spots , up-regulated in the gels of the attenuated strain , identified 32 spots as specific to the protozoan. Additionally, the analysis identified a number of E. coli DH5α proteins which were detected as differentially expressed by the computational gel image and statistical analysis.