Proteomic analysis is a powerful technology to enhance our insight into the pathogenesis, biomarkers and prevention of disease. Two-dimensional polyacrylamide gel electrophoresis (2-DE) is an important proteomics tool, where thousands of protein spots can be visualized, resulting in a global view of the state of a proteome. Viral infection will modify the patterns of host cell protein expression, which can affect the normal physiological function of host cell and determine viral pathogenic progress and consequence. To uncover host cellular responses in the early stage of classical swine fever virus infection, a proteomic analysis was conducted using 2DE followed by MALDI-TOF-TOF identification. Altered expression of 21 protein spots in infected pk-15 cells at 24 h p.i. were identified in 2D gels, with 13 of these being characterized by MALDI-TOF-MS/MS. These proteins function in cytoskeletal, energy metabolism, nucleic acid/processing, and cellular stress. The expression alteration of these proteins presents the changes in physiological functions of host cells and provides a clue for further understanding of the mechanisms of CSFV infection and pathogenesis.