In early embryos of Ciona, an invertebrate chordate, the animal-vegetal axis is established by the combinatorial actions of maternal factors. One target of these maternal factors, Foxd, is specifically expressed in the vegetal hemisphere and stabilizes the animal-vegetal axis by activating vegetal hemisphere-specific genes and repressing animal hemisphere-specific genes. This dual functionality is essential for the embryogenesis of early ascidian embryos; however, the mechanism by which Foxd can act as both a repressor and an activator is unknown. Here, we identify a Foxd binding site upstream of Lhx3/4, which is activated by Foxd, and compare it with a repressive Foxd binding site upstream of Dmrt.a. We found that activating sites bind Foxd with low affinity while repressive sites bind Foxd with high affinity. Reporter assays confirm that this qualitative difference between activating and repressive Foxd binding sites is sufficient to change Foxd functionality. We therefore conclude that the outcome of Foxd transcriptional regulation is encoded in cis-regulatory elements.