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The molecular mechanisms underlying OPC differentiation remain largely unknown. Here, we show that the thirty\u2010kDa HIV\u20101 Tat interacting protein (TIP30) is a negative regulator in oligodendrocyte development. The TIP30<jats:sup>\u2212\/\u2212<\/jats:sup> mice displayed an increased myelin protein level at postnatal day 14 and 21. By using a primary OPC culture system, we demonstrated that overexpression of TIP30 dramatically inhibited the stage progression of differentiating OPCs, while knockdown of TIP30 enhanced the differentiation of oligodendroglial cells remarkably. Moreover, overexpression of TIP30 was found to sequester the transcription factor Olig1 in the cytoplasm and weaken its nuclear translocation due to the interaction between TIP30 and Olig1, whereas knockdown of TIP30 led to more Olig1 localized in the nucleus in the initiation stage during OPC differentiation. In the cuprizone\u2010induced demyelination model, there was a dramatic increase in NG2\u2010expressing cells with nuclear location of Olig1 in the corpus callosum during remyelination. In contrast, within chronic demyelinated lesions in multiple sclerosis, TIP30 was abnormally expressed in NG2\u2010expressing cells, and few nuclear Olig1 was observed in these cells. Taken together, our findings suggest that TIP30 plays a negative regulatory role in oligodendroglial differentiation. 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