ST cells are known to be sensitive to TGEV, resulting in increased viral multiplication and CPEs. In order to better understand the interactions between the host proteome and TGEV, we adopted an iTRAQ quantitative proteomic approach to investigate the altered cellular proteins of the ST cells during TGEV infection in vitro. Compared with the 2-DE and 2D-DIGE methods often used, the 2D-LC-MS/MS method utilized here provides more quantitative and qualitative information about the proteins, and can also detect membrane proteins, hydrophobic proteins, higher molecular weight proteins, and low-abundance proteins,Mycophenolic acid which are often missed by other methods. iTRAQ also has more advantages compared to isotope-coded affinity tags and stable isotope labeling by amino acids in cell culture methods, which both allow multiple labeling and quantitation of four to eight samples simultaneously with high sensitivity. Further, the iTRAQ technique has been widely used for quantitative proteomics, including protein expression analysis and biomarker identification. Prior to proteomic analysis, we determined which time points to investigate following infection by observing the morphological changes and analyzing viral gene expression dynamics in the TGEV infected cells. The results indicated that TGEV induced significant CPEs from 40 to 64 hpi in infected cells compared to the mock infected cells. At 40 hpi,Phenoxybenzamine hydrochloride less than 50% of the infected cells were morphologically altered, while at 48 hpi more than 80% infected cells showed rounding and granular degeneration. Further, the mRNA level of the viral N gene in ST cells continuously increased in the infected cells until 48 h, at which time we observed the highest viral replication level. At 64 hpi, the morphological effects observed were much more pronounced, characterized by even more cellular rounding and detachment. However, the mRNA levels of the viral N gene decreased rapidly from 48 to 64 h, a phenomenon we believe may be attributed to the host’s immune response or a decrease in infected cell viability as the TGEV infection progressed. Based on our qRT-PCR and CPE analyses, we choose to more deeply investigate the proteomic changes occurring in the TGEV-infected ST cells at 48 hpi and 64 hpi using a 4-plex iTRAQ analysis.