Objective To clone and express Toxoplasma gondii gliding-associated protein 45 (TgGAP45) as recombinant protein and analyze its immunoreactivity by T. gondii infected human serum for its potential as an immunodiagnostic antigen. Methods Total RNA was extracted from tachyzoites of T. gondii RH strain and reversely transcribed into cDNA (RT-PCR). The DNA encoding for the open reading frame of TgGAP45 was amplified with gene-specific primers based on TgGAP45 sequence(GenBank accession No. AF453384.1). The PCR products were cloned into bacterial expression vector pET-30a(+) using EcoRⅠ and XhoⅠ sites. The correct recombinant plasmid pET-30a(+)-TgGAP45 was identified by PCR with gene-specific primers, double restriction enzyme digestion and DNA sequencing. The pET-30a(+)-TgGAP45 plasmid DNA was transformed into E. coli Rosetta (DE3) and the recombinant TgGAP45 protein (rTgGAP45) was expressed under induction of IPTG. The rTgGAP45 was purified with Ni-NTA column and analyzed by SDS-PAGE for its purity and Western blotting for its immune-recognition by T. gondii infected human serum and mouse anti-T. gondii soluble tachyzoites antigen (STAg) serum. Results DNA coding for TgGAP45 was successfully amplified from T. gondii tachyzoites total RNA by RT-PCR as 750 bp in length, then cloned into bacterial expression vector pET-30a(+). The correct recombinant plasmid pET-30a(+)-TgGAP45 was identified by gene-specific PCR, double restrict enzyme digestion and DNA sequencing. The TgGAP45 was successfully expressed as soluble recombinant protein in E. coli BL21 (DE3) under induction of IPTG with a rough molecular weight of 35 000, and purified with Ni-NTA. The His-tagged rTgGAP45 was strongly recognized not only by the anti-His antibody, but also by T. gondii-infected human serum and mouse anti-T. gondii STAg serum by Western blotting. Conclusion DNA encoding for TgGAP45 was successfully cloned from T. gondii tachyzoites and rTgGAP45 was efficiently expressed as soluble protein in E.coli BL21 (DE3). The purified rTgGAP45 remained its immunoreactivity to T. gondii infected human serum and mouse immune sera.