Abstract: Microbial phospholipase D (PLD) showed a more competent potential for phospholipids production due to its higher catalytic activity and broader substrate specificity. In this study, the PLD from Acinetobacter sp. DUT-2 (ADPLD) was used as the research object. Firstly, bioinformatics was used to examine the protein sequence characteristics. Then, the recombinant plasmid was generated and heterologously expressed in Escherichia coli. The enzyme protein was purified further, and the substrate selectivity of ADPLD to phosphatidylcholine (PC) with various acyl chains was investigated. Finally, the substrate recognition mechanism of ADPLD was investigated using molecular docking and molecular dynamics simulation. Multiple sequence alignment and phylogenetic tree analysis of ADPLD and other microbial-derived PLDs revealed that the sequence similarity between ADPLD and Streptomyces-derived PLDs was less than 30%, and there was only one conserved HKD motif, indicating that the catalytic mechanism of ADPLD might differ from the reaction mechanism in traditional cognition, which required two HKD motifs to complete the PLD catalytic process. ADPLD was mostly produced as a soluble protein, and a relatively uniform protein could be purified using Ni²⁺ affinity chromatography at a low concentration of 50 mmol/L imidazole. When soybean PC was utilized as a substrate, the specific activity of ADPLD was about 4.09 U/mg. ADPLD showed relatively high activity in neutralizing short-chain PC (C6-C14), with a specific activity of 13.2 U/mg for 8: 0/8: 0-PC, which was higher than that of other PC substrates with long acyl chains. The activity of ADPLD on PC reduced dramatically when the acyl chain length of PC grew from C14 to C16. Molecular dynamics simulation and molecular docking experiments revealed that the ADPLD amino acid residues Thr205, Pro209, Phe293, Ala324, Lys329, and Phe453 might form hydrophobic interactions with PC. Arg383 and Gly326 could form hydrogen bonds with PC when the distance between Arg383 (N), Gly326 (N), and PC (P) was <3 Å. These results indicate that ADPLD can form a stable enzyme substrate intermediate with phospholipid molecules. These findings set the groundwork for ADPLD molecular modification and future industrial applications.