Infection with reticuloendotheliosis virus (REV), a gammaretrovirus in the family Retroviridae, can result in immunosuppression and subsequent increased susceptibility to secondary infections. In the present study, we identified differentially expressed proteins in the spleens of chickens infected with the REV-A HLJ07I strain, using two-dimensional gel electrophoresis on samples from time points coinciding with different phases of the REV life cycle. Differentially expressed proteins were identified using one-dimensional liquid chromatography electrospray ionization tandem mass spectrometry (1D LC ESI MS/MS). Comparative analysis of multiple gels revealed that the majority of changes occurred at early stages of infection. In total, 60 protein spots representing 28 host proteins were detected as either quantitatively (false discovery rate [FDR] ≤0.05 and fold change ≥2) or qualitatively differentially expressed at least once during different sampling points. The differentially expressed proteins identified in this study included antioxidants, molecular chaperones, cellular metabolism, formation of the cytoskeleton, signal transduction, cell proliferation and cellar aging. The present findings provide a basis for further studies to elucidate the role of these proteins in REV-host interactions. This could lead to a better understanding of REV infection mechanisms that cause immune suppression.