The transient absorption spectroscopy of hydrolysis of the chromogenic substrate nitrocefin by the L1 metallo-β-lactamase (MβL), a bacterial enzyme responsible for destruction of β-lactam antibiotics molecules, showed formation and decay of a plausible red-shifted reaction intermediate. We propose a mechanism of this important reaction consistent with the transient kinetic data. Quantum mechanics/molecular mechanics (QM/MM) simulations of the reaction pathway revealed occurrence of two reaction intermediates (I1, I2) between the enzyme-substrate (ES) and enzyme-product (EP) complexes. The vertical S0-S1 transition energies calculated at the minimum energy structures (ES, I1, I2, EP) using the time dependent DFT (TD-DFT) method allowed us to assign the experimental absorption bands to all reacting species. We numerically solved the equations of chemical kinetics with the rate constants of all elementary steps evaluated with the transition state theory and simulated the kinetic curves as well as the evolution of the absorption bands of ES, I2, and EP. Direct comparison to the experimental data allowed us to identify the I2 intermediate as the transient red-shifted species detected experimentally. In agreement with the experimental observations, the recomputed energy profiles for the D120N and D120C mutants of L1 reacting with nitrocefin showed absence of a stable intermediate I2. According to the consistent experimental and theoretical results, the breakdown of the intermediate I2 corresponds to the rate-limiting stage of the chemical transformations in the active site of the L1 metallo-β-lactamase. On this basis, we established a QSAR-type correlation between the observed reaction rates (kcat) of three cephalosporin antibiotics (cefotixin, nitrocefin, cefepime) showing different hydrolysis rates by the L1 metallo-β-lactamase and different structures of the corresponding intermediates of the I2 type. This correlation can be employed for a rational design of novel antibiotics, which are not decomposed by metallo-β-lactamases.