Serial x-ray crystallography can uncover binding events, and subsequent chemical conversions occurring during enzymatic reaction. Here we reveal the structure, binding and cleavage of moxalactam antibiotic bound to L1 metallo-β-lactamase (MBL). Using time-resolved serial synchrotron crystallography we showed the time course of β-lactam hydrolysis and assembled molecular movie spanning 4 seconds. The reaction was initiated by laser pulse releasing Zn2+ ions from a UV-labile photocage. Two metal ions bind to the active site, followed by binding of moxalactam and the intact β-lactam ring is observed for 100 ms after photolysis. Cleavage of β-lactam is detected at 150 ms and the ligand is significantly displaced. The reaction product adjusts its conformation reaching steady state at 2000 ms corresponding to the relaxed state of the enzyme. Only small changes are observed in the positions of Zn2+ ions and the active site residues. Mechanistic details captured here should be generalized to other MBLs.