The anterior-posterior (A-P) axis in ascidian embryos is established through the posteriorizing activities of a localized egg region known as the posterior vegetal cortex/cytoplasm (PVC). Here we describe a novel function of macho-1, a maternally-localized muscle determinant, in establishment of the A-P axis in the Halocynthia roretzi embryo. Macho-1, in addition to its known function in the formation of posterior tissue such as muscle and mesenchyme, and suppression of the anterior-derived notochord fate, acts independently of its transcriptional activity as a regulator of posterior-specific unequal cell divisions, in cooperation with beta-catenin. Our results suggest that macho-1 and beta-catenin regulate the formation of a microtubule bundle that shortens and pulls the centrosome toward a sub-cellular cortical structure known as centrosome-attracting body (CAB), which is located at the posterior pole of the embryo during unequal cell divisions, and act upstream of PEM, a recently-identified regulator of unequal cell divisions. We also present data that suggest that PEM localization to the CAB may not be required for unequal cleavage regulation. The present study provides an important and novel insight into the role of the zinc-finger-containing transcription factor and indicates that it constitutes a major part of the PVC activity.