Multiple sclerosis (MS) is a demyelinating disease of the central nervous system (CNS). No effective remyelination therapies are in use. We hypothesized that thymosin beta4 (Tβ4) is an effective remyelination treatment by promoting differentiation of oligodendrocyte progenitor cells (OPCs), and that the epidermal growth factor receptor (EGFR) signaling pathway contributes to this process. Two demyelination animal models were employed in this study: 1) experimental autoimmune encephalomyelitis (EAE), an animal model of MS. EAE mice were treated daily for 30days, with Tβ4 or saline treatment initiated on the day of EAE onset; and 2) cuprizone diet model, a non-inflammatory demyelination model. The mice were treated daily for 4weeks with Tβ4 or saline after fed a cuprizone diet for 5weeks. Immunofluorescent staining and Western blot were performed to measure the differentiation of OPCs, myelin and axons, respectively. To obtain insight into mechanisms of action, the expression and activation of the EGFR pathway was measured. AG1478, an EGFR inhibitor, was employed in a loss-of-function study. Data revealed that animals in both demyelination models exhibited significant reduction of myelin basic protein (MBP(+)) levels and CNPase(+) oligodendrocytes. Treatment of EAE mice with Tβ4 significantly improved neurological outcome. Double immunofluorescent staining showed that Tβ4 significantly increased the number of newly generated oligodendrocytes identified by BrdU(+)/CNPase(+) cells and MBP(+) mature oligodendrocytes, and reduced axonal damage in the EAE mice compared with the saline treatment. The newly generated mature oligodendrocytes remyelinated axons, and the increased mature oligodendrocytes significantly correlated with functional improvement (r=0.73, p<0.05). Western blot analysis revealed that Tβ4 treatment increased expression and activation of the EGFR pathway. In the cuprizone demyelination model, Tβ4 treatment was confirmed that significantly increased OPC differentiation and remyelination, and increased the expression of EGFR and activated the EGFR pathway in the demyelinating corpus callosum. In cultured OPCs, blockage of the activation of the EGFR pathway with AG1478 abolished the Tβ4-increased OPC differentiation. Collectively, these findings indicate that: 1) Tβ4 increases proliferation of OPCs and the maturation of OPCs to myelinating oligodendrocytes which in concert, likely contribute to the beneficial effect of Tβ4 on EAE, 2) EGFR upregulated and activated by Tβ4 may mediate the process of OPC differentiation, and 3) Tβ4 could potentially be developed as a therapy for MS patients, and for other demyelinating neurological disorders.