Viral infections usually result in alterations in the host cell proteome which determine the fate of the infected cells and the progress of pathogenesis. To uncover cellular protein responses in classical swine fever virus-infected PK-15 cells, a proteomic analysis was conducted using 2D PAGE followed by MALDI-TOF-MS/MS identification. Altered expression of 35 protein spots in infected cells at 48 h p.i. were identified in 2D gels, with 21 of these being characterized by MALDI-TOF-MS/MS, including 16 upregulated proteins and 5 down-regulated proteins. Western-blot analysis confirmed the up-regulation of annexin 2 and down-regulation of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The altered proteins could be sorted into 7 groups according to cellular function: cytoskeleton, energy metabolism, replication/transcription and translation processes, protein processing, antioxidative stress proteins, heat shock proteins and signal transduction. The altered expression of these proteins provides a response profile of PK-15 host cells to CSFV infection. Further study of these altered proteins may facilitate understanding the mechanisms of CSFV infection and pathogenesis.