Abstract Rhomboid protein in Apicomplexa was associatedwith the process of host cell invasion. To evaluate the potential of the protein in eliciting protective immunityagainst challenge, a DNA vaccine pVAX1-Rho encodingEimeria tenella rhomboid was constructed. Recombinantprotein was expressed in Hela cells and verified by indirectimmunofluorescence and western blotting analysis. In vivoexperiments, 1-week-old chickens were randomly dividedinto three groups. Experimental group of chickens wereimmunized with DNA vaccines while control group ofchickens were injected with pVAX1 plasmid alone or sterilewater. Two weeks following the booster dose, all chickenswere inoculated orally with 5×104 sporulated oocysts of E.tenella. The host immunity and protective efficacy of thisvaccine against E. tenella challenge in broilers were evaluated.Results showed that specific antibody, the levels ofinterleukin-2 (IL-2), interferon-γ (IFN-γ), and the percentagesof CD4+ and CD8+ T lymphocyte cells were significantlyincreased in the pVAX1-Rho group. Challenge experimentsdemonstrated that pVAX1-Rho vaccination could reduceoocyst excretion, decrease cecal lesion, increase bodyweightgains and provide chickens with oocysts decreaseratio around 75.8 %. These results suggest that thepVAX1-Rho was able to induce humoral and cellularresponses and generate protective immunity against E.tenella infection.