Simple SummarySkeletal muscle is the most abundant tissue in animals, accounting for 45–60% of the body weight of meat animals, and the growth and development of skeletal muscle has the most direct impact on lean meat percentage and muscle quality. Therefore, exploring the molecular regulation mechanism of skeletal muscle growth and development is an important basis for improving pig meat production performance, and it has also been a research focus in animal genetics in recent years. In this study, ChIP-seq and other methods were used to explore the transcriptional regulation mechanism of new transcription factorMAMSTRin pig skeletal muscle development, The results of this study can further reveal the regulatory mechanism of pig skeletal muscle growth and development, identify new genes related to pig skeletal muscle growth and development, and provide a better reference for pig-farming practices and breeding of high-quality meat pig breeds.AbstractMyocyte enhancer factor-2-activating motif and SAP domain-containing transcriptional regulator (MAMSTR) regulates its downstream through binding in its promoter regions. However, its molecular mechanism, particularly the DNA-binding sites, and coregulatory genes are quite unexplored. Therefore, to identify the genome-wide binding sites of theMAMSTRtranscription factors and their coregulatory genes, chromatin immunoprecipitation sequencing was carried out. The results showed thatMAMSTRwas associated with 1506 peaks, which were annotated as 962 different genes. Most of these genes were involved in transcriptional regulation, metabolic pathways, and cell development and differentiation, such as AMPK signaling pathway, TGF-beta signaling pathway, transcription coactivator activity, transcription coactivator binding, adipocytokine signaling pathway, fat digestion and absorption, skeletal muscle fiber development, and skeletal muscle cell differentiation. Lastly, the expression levels and transcriptional activities ofPID1,VTI1B,PRKAG1,ACSS2, andSLC28A3were screened and verified via functional markers and analysis. Overall, this study has increased our understanding of the regulatory mechanism ofMAMSTRduring skeletal muscle fibroblast development and provided a reference for analyzing muscle development mechanisms.