Infection with the Gram-negative species Neisseria gonorrhoeae leads to inflammation that is responsible for the disease symptoms of gonococcal urethritis, cervicitis, and pelvic inflammatory disease. During growth these bacteria release significant amounts of peptidoglycan (PG) fragments which elicit inflammatory responses in the human host. To better understand the mechanisms involved in PG synthesis and breakdown in N. gonorrhoeae, we characterized the effects of mutation of mltG. MltG has been identified in other bacterial species as a terminase that stops PG strand growth by cleaving the growing glycan. Mutation of mltG in N. gonorrhoeae did not affect bacterial growth rate but resulted in increased PG turnover, more cells of large size, decreased autolysis under non-growth conditions, and increased sensitivity to antibiotics that affect PG crosslinking. An mltG mutant released greatly increased amounts of PG monomers, PG dimers, and larger oligomers. In the mltG background, mutation of either ltgA or ltgD, encoding the lytic transglycosylases responsible for PG monomer liberation, resulted in wild-type levels of PG monomer release. Bacterial two-hybrid assays identified positive interactions of MltG with synthetic penicillin-binding proteins PBP1 and PBP2 and the PG-degrading endopeptidase PBP4 (PbpG). These data are consistent with MltG acting as a terminase in N. gonorrhoeae and suggest that absence of MltG activity results in excessive PG growth and extra PG in the sacculus that must be degraded by lytic transglycosylases including LtgA and LtgD. Furthermore, absence of MltG causes a cell wall defect that is manifested as large cell size and antibiotic sensitivity.