Avian coccidiosis, which is characterized by intestinal damage in the host, is caused by the intracellular protozoan of Eimeria species, particularly Eimeria tenella. During its developmental cycle, various transitional stages are undergone by E. tenella, including oocysts, sporocysts, merozoites, and gametocytes. These transitions need both transcriptional and translational regulation, as well as post-translational protein modification. Myristoylation is a is a post-translational modification (PTM) of proteins that acts as a regulatory mechanism in signal transduction, cellular localization, and even oncogenesis. Previous studies showed that protein myristoylation is crucial for Plasmodium falciparum, Leishmania donovani, Trypanosoma brucei and Toxoplasma gondii. However, little information has been found on enzymes related to myristoylation or the role of protein myristoylation in E. tenella. Therefore, the myristoylation of the second-generation E. tenella merozoite was explored. In this study, we investigated the myristoylated proteome of E. tenella using chemoproteomic methods. We identified 104 proteins that involved in host cell invasion, motility, parasite morphology as well as ribosome components. In addition, proteasome component, electron transport chain and carbohydrate metabolism were identified. This study revealed the significant impact of myristoylation on E. tenella biology and pathogenicity. In brief, this study lays the foundation for future in-depth analysis and functional validation of myristoylated proteome in the life cycle of the avian parasite.