Adult skeletal muscles can regenerate after injury, due to the presence of satellite cells, a quiescent population of myogenic progenitor cells. Once activated, satellite cells repair the muscle damage by undergoing myogenic differentiation. The myogenic regulatory factors (MRFs) coordinate the process of progenitor differentiation in cooperation with other families of transcription factors (TFs). The Six1 and Six4 homeodomain TFs are expressed in developing and adult muscle and Six1 is critical for embryonic and adult myogenesis. However, the lack of a muscle developmental phenotype in Six4‐null mice, which has been attributed to compensation by other Six family members, has discouraged further assessment of the role of Six4 during adult muscle regeneration. By employing genome‐wide approaches to address the function of Six4 during adult skeletal myogenesis, we have identified a core set of muscle genes coordinately regulated in adult muscle precursors by Six4 and the MRF MyoD. Throughout the genome of differentiating adult myoblasts, the cooperation between Six4 and MyoD is associated with chromatin repressive mark removal by Utx, a demethylase of histone H3 trimethylated at lysine 27. Among the genes coordinately regulated by Six4 and MyoD are several genes critical for proper in vivo muscle regeneration, implicating a role of Six4 in this process. Using in vivo RNA interference of Six4, we expose an uncompensated function of this TF during muscle regeneration. Together, our results reveal a role for Six4 during adult muscle regeneration and suggest a widespread mechanism of cooperation between Six4 and MyoD.—Chakroun, I., Yang, D., Girgis, J., Gunasekharan, A., Phenix, H., Kærn, M., Blais, A. Genome‐wide association between Six4, MyoD, and the histone demethylase Utx during myogenesis. FASEB J. 29, 4738‐4755 (2015). www.fasebj.org