Mycobacterium avium subsp hominissuis (M. avium) is a pathogen that infects and survives in macrophages. Previously, we have identified the M. avium MAV_2941 gene encoding a 73 amino acid protein exported by the oligopeptide transporter OppA to the macrophage cytoplasm. Mutations in MAV_2941 were associated with significant impairment of M. avium growth in THP-1 macrophages. In this study, we investigated the molecular mechanism of MAV_2941 action and demonstrated that MAV_2941 interacts with the vesicle trafficking proteins syntaxin-8 (STX8), adaptor-related protein complex 3 (AP-3) complex subunit beta-1 (AP3B1) and Archain 1 (ARCN1) in mononuclear phagocytic cells. Sequencing analysis revealed that the binding site of MAV_2941 is structurally homologous to the human phosphatidylinositol 3-kinase (PI3K) chiefly in the region recognized by vesicle trafficking proteins. The β3A subunit of AP-3, encoded by AP3B1, is essential for trafficking cargo proteins, including lysosomal-associated membrane protein 1 (LAMP-1), to the phagosome and lysosome-related organelles. Here, we show that while the heat-killed M. avium when ingested by macrophages co-localizes with LAMP-1 protein, transfection of MAV_2941 in macrophages results in significant decrease of LAMP-1 co-localization with the heat-killed M. avium phagosomes. Mutated MAV_2941, where the amino acids homologous to the binding region of PI3K were changed, failed to interact with trafficking proteins. Inactivation of the AP3B1 gene led to alteration in the trafficking of LAMP-1. These results suggest that M. avium MAV_2941 interferes with the protein trafficking within macrophages altering the maturation of phagosome.