Abstract: In order to explore the structural and functional properties of the extracellular polysaccharide flippase Wzx from Lactobacillus fermentum A51, this paper systematically investigates the Wzx protein using bioinformatics technology. Firstly, the evolutionary characteristics of the Wzx protein are analyzed through multiple sequence alignment and the construction of a phylogenetic tree. Subsequently, the physicochemical properties, subcellular localization, functional domains, hydrophilicity/hydrophobicity, signal peptides, transmembrane regions, phosphorylation modification sites, glycosylation modification sites, advanced structure, and mode of action are predicted and analyzed. The results indicated that Wzx is composed of 472 amino acids, with a relative molecular mass of 53.31 kDa and a total atom count of 7651. The theoretical isoelectric point is 9.50, and the instability coefficient is 33.79, and it was an alkaline stable hydrophobic protein. Wzx lacks a signal peptide and functions as a flipping enzyme within the cell membrane. Following transmembrane domain prediction, it was determined that Wzx contains 14 transmembrane helix structures. A total of 33 significant phosphorylation sites were identified, comprising 14 serine (Ser) sites, 12 threonine (Thr) sites, and 7 tyrosine (Tyr) sites. Regarding subcellular localization and functional domain prediction, Wzx was predominantly localized in the endoplasmic reticulum (55.6%), indicating its membership in the Multidrug and Toxic Compound Extrusion family. In terms of the secondary structure of Wzx, α-helix constituted 61.44%, random coil accounted for 22.25%, and extended chain represented 16.31%, Based on the “V” shape of the three-dimensional structure of Wzx, it is speculated that it realizes the transport of extracellular polysaccharides through its own conformational reversal or its own conformational change. This study offers a theoretical foundation for a deeper understanding of the functional characteristics and mechanisms of action of Wzx, which is crucial for comprehending the transport processes of extracellular polysaccharides.