Satellite cells are stem cells that are critical for the formation and growth of skeletal muscle during myogenesis. To differentiate and fuse, proliferating satellite cells or myoblasts must migrate and establish stable cell-cell contacts. However, the factors that regulate myoblast migration and fusion are not understood completely. We have identified PGI₂ as a novel regulator of myogenesis in vitro. PGI₂ is a member of the family of prostaglandins (PG), autocrine/paracrine signaling molecules synthesized via the cyclooxygenase-1 and -2 pathways. Primary mouse muscle cells both secrete PGI₂ and express the PGI₂ receptor, IP, at various stages of myogenesis. Using genetic and pharmacological approaches, we show that PGI₂ is a negative regulator of myoblast migration that also enhances cell fusion. Thus, PGI₂ may act as a "brake" on migrating cells to facilitate cell-cell contact and fusion. Together, our results highlight the importance of the balance between positive and negative regulators in cell migration and myogenesis. This work may have implications for migration of other populations of adult stem cells and/or cells that undergo fusion.--Bondesen, B. A., Jones, K. A., Glasgow, W. C., Pavlath, G. K. Inhibition of myoblast migration by prostacyclin is associated with enhanced cell fusion.