Abstract: Adenylosuccinate synthase (Adss) is an essential enzyme in the purine biosynthesis process. Since it was found in Escherichia coli in 1956, Adss has been reported about 30000 different gene sequences in NCBI. It was found in various species from bacteria to mammals, reflects a high level of genetic diversity. This gene plays an important role in the metabolism of nucleotide cycles. Therefore, it has been used as a target molecule for the researches of various drugs. In the study of the mechanism of hEGF action on asexual reproduction of Stylonychia lemnae, an unicellular animal model, Adss gene was found to be possibly involved in the regulatory process of cell division and cell cycle. In order to further develop the functional and molecular mechanism of Adss gene in protozoa, we cloned the Adss gene of Stylonychia lemnae by PCR. The sequence structure, basic physical and chemical properties and conserved domain of the protein were analyzed. The ML phylogenetic tree based on Adss amino acid sequences of different organisms was illustrated. Furthermore, the expression and localization vector of Adss gene was constructed by double enzyme digestion of Adss cDNA and pEGFP-N1 vector to confirm the subcellular localization of Adss protein with fluorescence staining. The results showed that the total length of the S. lemnae Adss gene is about 1396 bp, encoding 458 amino acids. Its conserved domain containes a P-loop NTPase. Tertiary structure prediction suggested that Adss protein is composed of α-helix, β-sheet and random coil. The Adss gene of S. lemnae has high homology and close genetic relationship with Paramecium tetraurelia, Ichthyophthirius multifiliis and Tetrahymena thermophila according to multiple sequence alignment and phylogenetic analysis. With a promoter sequence-regulated expression vector pEGFP-N1-Adss, the S. lemnae Adss protein mainly distribute in the cytoplasm. These results provide many valuable data and basics for the research on the function and molecular mechanism of protozoan Adss gene.