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Long noncoding RNAs have been identified as important regulators of gene expression and animal development. The expression of natural antisense transcripts (NATs) transcribed in the opposite direction to protein-coding genes is usually positively correlated with the expression of homologous sense genes and is the key factor for expression. Here, we identified a conserved noncoding antisense transcript, CFL1-AS1, that plays an important role in muscle growth and development. CFL1-AS1 overexpression and knockout vectors were constructed and transfected into 293T and C2C12 cells. CFL1-AS1 positively regulated CFL1 gene expression, and the expression of CFL2 was also downregulated when CFL1-AS1 was knocked down. CFL1-AS1 promoted cell proliferation, inhibited apoptosis and participated in autophagy. This study expands the research on NATs in cattle and lays a foundation for the study of the biological function of bovine CFL1 and its natural antisense chain transcript CFL1-AS1 in bovine skeletal muscle development. The discovery of this NAT can provide a reference for subsequent genetic breeding and data on the characteristics and functional mechanisms of NATs.