Objective To prepare rabbit-antimouse testis expressed 33 (Tex33) polyclonal antibody and detect the expression of Tex33 during mouse spermatogenesis. Methods Tex33 open reading frame was amplified by PCR from mouse testis cDNA. The PCR product was finally sub-cloned into pET-30a vector. The prokaryotic expression plasmid pET-30a-Tex33 was constructed and then transformed into E. coli BL21. Tex33 prokaryotic protein was induced by IPTG and then purified by Ni-NTA affinity chromatography. Tex33 polyclonal antibody was obtained from adult male New Zealand white rabbits immunized with purified Tex33 protein. ELISA, Western blotting and immunofluorescence assays were used to determine the potency and specificity. Results pET-30a-Tex33 recombinant vector was successfully constructed. After induced by IPTG, the recombinant Tex33 protein could be expressed effectively. The titer of polyclonal antibody was 1:1 000 000. Western blotting showed that the antibody could recognize Tex33 protein in mouse testis. Immunofluorescence assay revealed that Tex33 gene was expressed in spermatids and sperms of adult mouse testis. And Tex33 was located on the acrosome and flagellum of spermatozoa. Conclusion We have prepared rabbit-antimouse Tex33 polyclonal antibody with high specificity and found that Tex33 gene was expressed in mouse testis.