In bacteria, the acyl transmembrane modification of cell-surface polymers is a common feature to strengthen their pathogenic potential. The dlt operon-mediated D-alanylation of lipoteichoic acids (LTAs) is an important post-modification to adjust charge balance in Gram-positive bacteria. Four proteins of DltA/B/C/D were identified to be essential for LTA D-alanine incorporation. Though the process of D-alanine transfer by cytoplasmic DltA/DltC has been largely probed, transmembrane catalysis by MBOAT protein DltB and the terminal player DltD is yet to be defined. Here, the crystal structure of stDltD was determined from S. thermophilus at 2.94 ? resolution. On the basis of the structure comparison, DltD was considered as the terminal acyltransferase of the dlt operon, and it belonged to the SGNH-like family. An stDltD active center, including four blocks and a catalytic triad, conservatively exists in various Gram-positive pathogens. In addition, structural analysis showed that the stDltD catalytic center formed a strong positively charged groove docking with a glycerolphosphate molecule. Combined with previous reports, an updated working model was proposed for cross-membrane D-alanylation mediated by the dlt operon. The structural evidence provides more implications to clarify the biological function of stDltD and the process of transmembrane acyl modification.