Gametocyte proteins of Eimeria spp. are essential components of the oocyst wall, and some of these proteins have been analysed to identify targets of transmission-blocking vaccines against avian coccidiosis. In the present study, a cDNA from E. necatrix gametocytes was cloned and sequenced. The cDNA is 1,473 bp in length and encodes a 490-amino-acid protein containing a tyrosine-serine (Tyr/Ser)-rich domain and a proline-methionine (Pro/Met)-rich domain. A quantitative real-time PCR (qPCR) analysis showed that the cDNA is expressed only during gametogenesis. A fragment containing the Tyr/Ser-rich domain (rEnGAM59) was expressed in Escherichia coli BL21 (DE3) cells. Immunoblotting showed that rEnGAM59 was recognized by the serum of convalescent chickens after infection with E. necatrix, and that an anti-rEnGAM59 antibody recognized a ～59 kDa protein and two other proteins (～35 kDa and ～33 kDa) in gametocyte extracts. An immunofluorescence assay showed that the anti-rEnGAM59 antibody recognized wall-forming bodies in the macrogametocytes and oocyst walls. An in vivo vaccination and challenge trial was conducted to test the potential utility of rEnGAM59 as a vaccine. Immunized chickens performed better than the unimmunized and challenged (positive control) chickens. The intestinal lesion scores were significantly lower in the immunized groups than in the positive control group (P < 0.05). In contrast, the body weight gains (BWG) were significantly higher in the immunized groups than in the positive control group (P < 0.05). There were no significant differences in the lesion scores and BWG between the groups immunized with rEnGAM59 protein or with live oocysts (P > 0.05). Chickens immunized with rEnGAM59 protein had a significantly higher antigen-specific serum IgY response (P < 0.05). rEnGAM59 protein can be used as candidate antigen to develop a recombinant coccidiosis vaccine.