Eimeria species are intracellular obligate parasites, among the most common pathogens affecting the intensive poultry industry. Oxidoreductases are members of a class of proteins with redox activity and are widely found in apicomplexan protozoans. However, there have been few reports related to Eimeria species. In this study, total RNA was extracted from the gametocytes of E. necatrix Yangzhou strain to amplify the EnOXIO1 gene using reverse-transcription polymerase chain reaction. After cloning and sequence analysis, the prokaryotic expression vector pET-28a(+)-EnOXIO1 was constructed and transformed into Escherichia coli BL21(DE3), and the recombinant protein rEnOXIO1 was expressed by induction with isopropyl ß-D-1-thiogalactopyranoside. The full length EnOXIO1 gene was 2535 bp encoding 844 amino acids, and the EnOXIO1 protein had a molecular weight of about 100 kDa and was mainly expressed in inclusion bodies. Western blot analysis indicated that the rEnOXIO1 protein had good antigenicity and cross-reactivity and was specifically recognized by a 6 ×HIS labeled monoclonal antibody, mouse anti-recombinant protein polyclonal antibody, and recovery serum from chickens infected with E. necatrix, E. acervulina, and E. tenella sporulated oocysts. The results of laser confocal immunofluorescence localization showed that the EnOXIO1 protein was mainly located on the wall-forming bodies in gametocytes and played an important role in the formation of the oocyst wall. Quantitative PCR analysis revealed that transcript levels of EnOXIO1 were highest in the gametocyte stage. Protein expression levels of EnOXIO1 were higher in the gametocyte stage than in other developmental stages according to western blot analysis. Vaccination of chickens against E. necatrix was achieved with recombinant protein rEnOXIO1, which triggered humoral immunity and antibody production, increased average body weight gain, reduced oocyst output and alleviated lesions after E. necatrix infection. The highest ACI value (172.36) was observed in chickens that received 200 μg rEnOXIO1 compared with other immunization groups.