Prion diseases or transmissible spongiform encephalopathies (TSEs) are fatal diseases associated with the conversion of the cellular prion protein (PrPC) to the abnormal prion protein (PrPSc). Since the molecular mechanisms in pathogenesis are widely unclear, we analyzed the global phospho-proteome and detected a differential pattern of tyrosine- and threonine phosphorylated proteins in PrPSc-replicating and pentosan polysulfate (PPS)-rescued N2a cells in two-dimensional gel electrophoresis. To quantify phosphorylated proteins, we performed a SILAC (stable isotope labeling by amino acids in cell culture) analysis and identified 105 proteins, which showed a regulated phosphorylation upon PrPSc infection. Among those proteins, we validated the dephosphorylation of stathmin and Cdc2 and the induced phosphorylation of cofilin in PrPSc-infected N2a cells in Western blot analyses. Our analysis showed for the first time a differentially regulated phospho-proteome in PrPSc infection, which could contribute to the establishment of novel protein markers and to the development of novel therapeutic intervention strategies in targeting prion-associated disease.