In this study, we detected the viral DNA of Human Herpes Virus 6 (HHV-6) in the sera and cell-free cerebrospinal fluid (CSF) of Chinese multiple sclerosis (MS) patients. The results revealed that the copy numbers of serum HHV-6 viral DNA were higher in MS than in normal subjects (NS) or in other neurologic diseases (OND). We also found that in the MS subjects, most T cells recognizing myelin basic protein (MBP) were cross-reactive and could be activated by a synthetic peptide corresponding to residues of HHV-6 or EBV. The estimated precursor frequency of these cross-reactive T cells recognizing both peptides, MBP and HHV-6 or EBV, was significantly elevated in MS compared with that in controls. More significant was the presence of CD8+ cytotoxic cross-reactive T cells, as they could directly induce injury to oligodendrocytes that are known to express both MBP and MHC class I molecules. The study provides important evidence for understanding the potential role of HHV-6 or EBV infection in the pathogenesis of MS.