To reveal the important roles of the ESAT-6 protein in the virulence, immune response, and cellular functions of Mycobacterium marinarius , this study employs bioinformatics methods to analyze the physicochemical properties, subcellular localization, protein structure, synteny, and protein-protein interaction network of the ESAT-6 protein from Mycobacterium marinarius , and to predict its functional roles. The results indicate that ESAT-6 protein is a hydrophilic protein composed of approximately 100 amino acids, serving as a T-cell antigen, is predominantly located in the nucleus and cytoplasm, and possesses a helical hairpin structure. Motif analysis reveals that the ESAT-6 protein contains three conserved motifs, and synteny analysis with Mycobacterium tuberculosis, Mycobacterium kansasii , and Mycobacterium ulcerans shows a high degree of conservation. The ESAT-6 protein directly interacts with proteins such as esxG, espA, and espB, and can also form a complex with CFP-10 protein to exert its functions. The functions of the ESAT-6 protein mainly involve the formation of specialized pores on the cell membrane to disrupt membrane, facilitate immune evasion, and induce cell apoptosis, etc., providing theoretical basis for studying the mechanism of Mycobacterium marinarius infection in hosts.