Human monoacylglycerol lipase (MAGL) is a membrane-interacting enzyme that generates pro-inflammatory signaling molecules. For this reason， MAGL inhibition is a promising strategy to treat pain， cancer， and neuroinflammatory diseases. MAGL can hydrolyze monoacylglycerols bearing an acyl chain of different lengths and degrees of unsaturation， cleaving primarily the endocannabinoid 2-arachidonoylglycerol. Importantly， the enzymatic binding site of MAGL is confined by a 75-amino-acid-long， flexible cap domain， named 'lid domain'， which is structurally similar to that found in several other lipases. However， it is unclear how lid domain plasticity affects catalysis in MAGL. By integrating extensive molecular dynamics simulations and free-energy calculations with mutagenesis and kinetic experiments， we here define a lid-domain-mediated mechanism for substrate selection and binding in MAGL catalysis. In particular， we clarify the key role of Phe159 and Ile179， two conserved residues within the lid domain， in regulating substrate specificity in MAGL. We conclude by proposing that other structurally related lipases may share this lid-domain-mediated mechanism for substrate specificity.