Avibactam is a beta-lactamase inhibitor that is in clinical development, combined with beta-lactam partners, for the treatment of bacterial infections comprising Gram-negative organisms. Avibactam is a structural class of inhibitor that does not contain a beta-lactam core but maintains the capacity to covalently acylate its beta-lactamase targets. Using the TEM-1 enzyme, we characterized avibactam inhibition by measuring the on-rate for acylation and the off-rate for deacylation. The deacylation off-rate was 0.045 min(-1), which allowed investigation of the deacylation route from TEM-1. Using NMR and MS, we showed that deacylation proceeds through regeneration of intact avibactam and not hydrolysis. Other than TEM-1, four additional clinically relevant beta-lactamases were shown to release intact avibactam after being acylated. We showed that avibactam is a covalent, slowly reversible inhibitor, which is a unique mechanism of inhibition among beta-lactamase inhibitors.