Objective To analyze the sequence of mouse spermatogenesis associated protein 3 ( Spata3) and explore its structure and function. Methods The NCBI database was used to compare the homology of mouse Spata3. ExPASy ProtParam software was used to analyze its physical and chemical characteristics. SOPMA and GOR4 predicted the spatial conformation. NetPhos3. 1 and the STRING database were used to analyze protein modification sites and protein- protein interactions. Immunohistochemistry and immunofluorescence were used to observe the localization of Spata3 protein. Results Mouse Spata3 had 64% homology with humans in coding sequence. It was a basic unstable hydrophilic protein without a signal peptide. It was a non-transmembrane intracellular protein. It was mainly located in the nucleus and cytoplasm of sperm at all levels of testicular tissue, and its expression in round sperm cells was the highest. Spata3 contained an internal disordered domain, 30 potential phosphorylation sites, and 11 potential O-glycosylation sites, which may interact with proteins such as Spata46 and Spert. Conclusions Spata3 is a conserved protein in spermatogenesis and may regulate spermatogenesis.