β-lactam antibiotics act as suicide substrates of transpeptidases responsible for the last cross-linking step of peptidoglycan synthesis in the bacterial cell wall. Nucleophilic attack of the β-lactam carbonyl by the catalytic residue (Ser or Cys) of transpeptidases results in the opening of the β-lactam ring and in the formation of a stable acyl-enzyme. The acylation reaction is considered as irreversible due to the strain of the β-lactam ring. In contradiction with this widely accepted but poorly demonstrated premise, we show here that the acylation of the L,D-transpeptidase Ldtfm from Enterococcus faecium by the β-lactam nitrocefin is reversible, leading to limited antibacterial activity. Experimentally, two independent methods based on spectrophotometry and mass spectrometry provided evidence that recyclization of the β-lactam ring within the active site of Ldtfm regenerates native nitrocefin. Ring strain is therefore not sufficient to account for irreversible acylation of peptidoglycan transpeptidases observed for most β-lactam antibiotics.