Tight regulation of Arp2/3 complex is required to allow productive nucleation of force-generating, branched actin networks. An emerging aspect of regulation is the incorporation of subunit isoforms into Arp2/3 complex. Specifically, both isoforms of the ArpC5 subunit, ArpC5 and ArpC5L, have been reported to fine-tune nucleation activity and branch junction stability. Elevated levels of ArpC5 have also been linked to increased cancer progression and metastasis. Here, we have combined genetic engineering of cells and cellular structural biology to describe how ArpC5 and ArpC5L differentially regulate cell migration. They do so by defining the structural stability of ArpC1 in branch junctions and, in turn, by determining protrusion characteristics, protein dynamics, and actin network ultrastructure. ArpC5 isoforms also have an impact on the positioning of actin assembly factors from the Ena/VASP family, which act downstream of Arp2/3 complex-mediated nucleation. This suggests that ArpC5 and Ena/VASP proteins, both predictors for poor outcome in cancer, are part of a signaling pathway enhancing cell migration and, by inference, metastasis.