BACKGROUND: Maduramicin ammonium (MA), a widely used coccidiostat, has been reported to cause skeletal muscle degeneration in animals and even humans. In this study, we explore the underlying mechanism of its toxicity in skeletal muscle. RESULTS: First, we observed that MA impaired autophagic flux which was evidenced by increased protein level of LC3-II and p62 in skeletal myoblast C2C12 and L6 cell lines and rectus femoris muscle tissues of rats and broilers. Then, we found that MA induced eIF2α phosphorylation and ATF4 expression in the cells and tissues. Co-treatment with ISRIB attenuated MA-induced LC3-II and p62 in C2C12 and L6 cells, suggesting that MA-induced eIF2α-ATF4 pathway contributed to impairment of autophagic flux in the cells. Lastly, we showed that MA activated AMPK signaling in skeletal muscle, since the phosphorylation of AMPK was increased by MA treatment in skeletal myoblast cell lines and muscle tissues. Furthermore, in AMPK downregulated C2C12 cells, MA-induced LC3-II, ATF4 and phosphorylation of eIF2α was reversed, supporting that AMPK was involved in the regulation of the eIF2α-ATF4 pathway and autophagic flux during MA exposure. CONCLUSION: Our findings showed that MA impairs autophagic flux through activating the AMPK-induced eIF2α-ATF4 pathway in skeletal muscle. © 2024 Society of Chemical Industry.