Actin cytoskeleton drives cell migration in muscle and non-muscle cells and plays a major role in such diverse processes as tissue morphogenesis, muscle contraction, and cancer metastases. In non-muscle cells, two major actin isoforms, beta and gamma actin, incorporate into different cytoskeletal structures and mediate cell migration and contractility. In our work we identified that beta, but not gamma actin, is possttranslationally arginylated, and that this arginylation is essential for normal cell migration. Differential arginylation of actin isoforms is regulated at the level of their nucleotide coding sequence. This regulation is a dynamic event that dynamically responds to cellular stimuli and is universal to multiple cell types. Thus, actin arginylation at the cell leading edge is a new regulatory process that acts at post-transcriptional and post-translational levels and is highly selective in targeting actin during cell migration.