Bacterial cells are surrounded by a peptidoglycan (PG) cell wall. This structure is essential for cell integrity and its biogenesis pathway is a key antibiotic target. Most bacteria utilize two types of synthases that polymerize glycan strands and crosslink them: class A penicillin-binding proteins (aPBPs) and complexes of SEDS proteins and class B PBPs (bPBPs). Although the enzymatic steps of PG synthesis are well characterized, the steps involved in terminating PG glycan polymerization remain poorly understood. A few years ago, the conserved lytic transglycosylase MltG was identified as a potential terminase for PG synthesis in Escherichia coli. However, characterization of the in vivo function of MltG was hampered by the lack of a growth or morphological phenotype in ΔmltG cells. Here, we report the isolation of MltG-defective mutants as suppressors of lethal deficits in either aPBP or SEDS/bPBP PG synthase activity. We used this phenotype to perform a domain-function analysis for MltG, which revealed that access to the inner membrane is important for its in vivo activity. Overall, our results support a model in which MltG functions as a terminase for both classes of PG synthases by cleaving PG glycans as they are being actively synthesized.