Prior to the somite segmentation, the cells in the anterior presomitic mesoderm (PSM) express a set of genes that is required for defining the segmental border and polarity of the prospective somite. However, little is known how the expression of these genes is repressed upon segmentation. Here we report that Bowline, an associate protein of the transcriptional corepressor XGrg-4, repressed Tbx6 dependent transcription of Thylacine1 by mediating interaction of Tbx6 with XGrg-4 in Xenopus laevis. In bowline-deficient embryos, segmental border formation was disturbed, and expression of Thylacine1, X-Delta-2, and bowline expanded anteriorly. Tbx6-dependent transcription of Thylacine1 was suppressed by Bowline, together with XGrg-4. We also found that Bowline mediated the interaction of Tbx6 and XGrg-4. Based on our findings, we conclude that a part of the transcriptional repression at the anterior end of the PSM is caused by Bowline mediated transcriptional repression of Tbx6-dependent gene expression in X. laevis.