In Gram-negative bacteria, the maintenance of lipid asymmetry (Mla) system is involved in the transport of phospholipids between the inner (IM) and outer membrane. The Mla system utilizes a unique IM-associated periplasmic solute-binding protein, MlaD, which possesses a conserved domain, MlaD domain. While proteins carrying the MlaD domain are known to be primarily involved in the trafficking of hydrophobic molecules, not much is known about this domain itself. Thus, in this study, the characterization of the MlaD domain employing bioinformatics analysis is reported. The profiling of the MlaD domain of different architectures reveals the abundance of glycine and hydrophobic residues and the lack of cysteine residues. The domain possesses a conserved N-terminal region and a well-preserved glycine residue that constitutes a consensus motif across different architectures. Phylogenetic analysis shows that the MlaD domain archetypes are evolutionarily closer and marked by the conservation of a functionally crucial pore loop located at the C-terminal region. The study also establishes the critical role of the domain-associated permeases and the driving forces governing the transport of hydrophobic molecules. This sheds sufficient light on the structure–function–evolutionary relationship of MlaD domain. The hexameric interface analysis reveals that the MlaD domain itself is not a sole player in the oligomerization of the proteins. Further, an operonic and interactome map analysis reveals that the Mla and the Mce systems are dependent on the structural homologs of the nuclear transport factor 2 superfamily.