Diclazuril has long been used as an effective benzeneacetonitrile anticoccidial for the control of Eimeria tenella that causes coccidiosis. However, the molecular mechanism underlying the anticoccidial effects of diclazuril remains elusive. In this study, a proteomic analysis of the effect of diclazuril on second-generation merozoites of E. tenella was performed. Using two-dimensional gel electrophoresis and real-time quantitative polymerase chain reaction (RT-PCR), 13 target proteins were found to be significantly affected by diclazuril treatment, with 11 of these proteins being identified as annotated proteins from E. tenella or other Apicomplexa parasites. These proteins contribute to various functions, including metabolism, protein synthesis, and host cell invasion. Using RT-PCR, we identified the potential pattern of transcriptional regulation induced by diclazuril, and we suggest some promising targets for the intervention of E. tenella infection.