Following myonecrosis, muscle satellite cells proliferate, differentiate and fuse, creating new myofibers. The Runx1 transcription factor is not expressed in naïve developing muscle or in adult muscle tissue. However, it is highly expressed in muscles exposed to myopathic damage yet, the role of Runx1 in muscle regeneration is completely unknown. Our study of Runx1 function in the muscle’s response to myonecrosis reveals that this transcription factor is activated and cooperates with the MyoD and AP-1/c-Jun transcription factors to drive the transcription program of muscle regeneration. Mice lacking dystrophin and muscle Runx1 (mdx - /Runx1 f/f), exhibit impaired muscle regeneration leading to age-dependent muscle waste, gradual decrease in motor capabilities and a shortened lifespan. Runx1-deficient primary myoblasts are arrested at cell cycle G1 and consequently differentiate. Such premature differentiation disrupts the myoblasts’ normal proliferation/differentiation balance, reduces the number and size of regenerating myofibers and impairs muscle regeneration. Our combined Runx1-dependent gene expression, ChIP-seq, ATAC-seq and histone H3K4me1/H3K27ac modification analyses revealed a subset of Runx1-regulated genes that are co-occupied by MyoD and c-Jun in mdx - /Runx1 f/f muscle. The data provide unique insights into the transcriptional program driving muscle regeneration and implicate Runx1 as an important participant in the pathology of muscle wasting diseases., Author Summary In response to muscle injury, the muscle initiates a repair process that calls for the proliferation of muscle stem cells, which differentiate and fuse to create the myofibers that regenerate the tissue. Maintaining the balance between myoblast proliferation and differentiation is crucial for proper regeneration, with disruption leading to impaired regeneration characteristic of muscle-wasting diseases. Our study highlights the important role the Runx1 transcription factor plays in muscle regeneration and in regulating the balance between muscle stem cell proliferation and differentiation. While not expressed in healthy muscle tissue, Runx1 level significantly increases in response to various types of muscle damage. This aligns with our finding that mice lacking Runx1 in their muscles suffer from impaired muscle regeneration. Their muscles contained a significantly low number of regenerating myofibers, which were also relatively smaller in size, resulting in loss of muscle mass and motor capabilities. Our results indicate that Runx1 regulates muscle regeneration by preventing premature differentiation of proliferating myoblasts, thereby facilitating the buildup of the myoblast pool required for proper regeneration. Through genome-wide gene-expression analysis we identify a set of Runx1-regulated genes responsible for muscle regeneration thereby implicating Runx1 in the pathology of muscle wasting diseases such as Duchenne muscular dystrophy.