The hydrolysis of carbapenem antibiotics by metallo-beta-lactamase enzymes (MBLs) is a biologically crucial reaction that promotes the antibiotic resistance, and consequently, MBLs cause human infections. Therefore, the enzymes that catalyze this reaction are among the most important pharmacological targets, especially those of the VIM type. Despite its relevance in the increase of antimicrobial resistance, the fundamental mechanism of meropenem (carbapenem antibiotic) hydrolysis catalyzed by this enzyme is not fully understood. Here, we report the catalytic mechanism of the meropenem hydrolysis by a VIM-1 metallo-beta-lactamase enzyme. We explored the chemical reaction with hybrid quantum mechanics/molecular mechanics (QM/MM) calculations, using three layers, two of them described by high-level ab initio methods at DLPNO-CCSD(T)/CBS plus M06-2X/6-311+G(2d,2p):AMBER. Our results demonstrate that the reaction occurs in three stages: nucleophilic addition, water orientation, and proton transfer. The rate-limiting step in the hydrolysis reaction was the initial stage with a Gibbs energy barrier of 15.7 kcal.mol(-1). This energy value is in excellent agreement with the experimental data of 15.9 kcal.mol(-1) (derived from the kcat value of 13 s(-1)). The Gibbs activation energy for the overall reaction was -14.5 kcal.mol(-1). Our biochemical understanding of the enzymatic regulation of meropenem hydrolysis by VIM-1 not only resolves the mechanism but also allows us to identify noncatalytic residues with an effect on the rate-limiting step of the reaction. That is, revisiting the electrostatic role of the residues in the second coordination sphere yields rationally identified mutants that can be used to inhibit the activity of the metallo-beta-lactamase enzyme or as a starting point for the design of ss-lactam antibiotics.