Tbx3, a member of T-box gene family, has been reported to play critical roles in embryonic development and cell fate determination. In mammalian tissues, Tbx3 is expressed as two isoforms called Tbx3 and Tbx3+2a. However, the differences between the two isoforms in pluripotency maintaining remain obscure. Here we show that both Tbx3 and Tbx3+2a are highly expressed in mouse embryonic stem cells (mESCs) or induced pluripotent stem cells (iPSCs). Overexpression of either Tbx3 or Tbx3+2a could induce the differentiation of mESCs. Mechanistic studies suggest both Tbx3 and Tbx3+2a inhibit the transcriptional activity of pluripotency related transcription factor Nanog. Moreover, Tbx3+2a could directly interact with Nanog while Tbx3 couldn't, indicating the requirement of the 2a domain in Nanog binding. In summary, our results not only reveal the essential roles of Tbx3 and Tbx3+2a in pluripotency maintaining, but also point out the different mechanistic modes between these two isoforms.