Abstract T-box factor, Tbx6, is a prerequisite for somite segmentation in vertebrates. We recently identified a negative regulator of Tbx6, Bowline, which represses the expression of genes involved in somite segmentation by suppressing the transcriptional activity of Tbx6. According to this function, bowline gene expression is restricted to the most anterior presomitic mesoderm where the somite segmentation program terminates, although it remains unclear how bowline expression is activated. To address this, we investigated the cis-regulatory region of bowline. Measuring luciferase activity driven by the bowline promoter, we found that Tbx6, Thylacine1, and E47 synergistically activate bowline expression in vitro. We also found that Tbx6, Thylacine1, and E47 are spatiotemporally sufficient to induce bowline expression in Xenopus somitogenesis. Our findings indicated that besides being a negative regulator of Tbx6, bowline itself is also regulated by Tbx6, suggesting the negative feedback loop of Tbx6-Bowline in the termination step of somite segmentation.