Anaplasma marginale is the rickettsia which causes the bovine anaplasmosis. The distribution of A. marginale is both tropical and subtropical regions of the world. The major surface protein 4 (MSP4) of this parasite was identified as an immunodominant protein. In this study, the full length of DNA encoding A. marginale MSP4 (AmMSP4) was cloned from the parasites. The open reading frame of msp4 coding sequence of Thailand strain is 849 bp. Phylogenetic analysis revealed that the msp4 coding sequence of A. marginale was highly conserved when compared with Anaplasma phagocytophilum. The recombinant plasmid was further transformed into the BL21-CodonPlus (DE3)-RIPL competent cells for over-expression of the recombinant major surface protein 4 of A. marginale (rAmMSP4). Sera from rabbit immunized with rAmMSP4 and from cattle infected with A. marginale were used to study the antigenicity of rAmMSP4 (35 kDa) and AmMSP4 (31 kDa). Both rAmMSP4 and AmMSP4 were recognized by these sera showing that recombinant and native AmMSP4 have conserved epitopes. Localization of Anaplasma parasites by immunofluorescence showed these parasites are distributed on both the membrane and the outside of infected erythrocytes. Regarding antigenicity, recombinant MSP4 could be used for immunodiagnostic purposes and as a possible vaccine candidate against anaplasmosis.