Understanding the molecular mechanism of inhibitor binding to prostate-specific membrane antigen (PSMA) is of fundamental importance for designing targeted drugs for prostate cancer. Here we designed a series of PSMA-targeting inhibitors with distinct molecular structures, which were synthesized and characterized using both experimental and computational approaches. Microsecond molecular dynamics simulations revealed the structural and thermodynamic details of PSMA-inhibitor interactions. Our findings emphasize the pivotal role of the inhibitor's P1 region in modulating binding affinity and selectivity and shed light on the binding-induced conformational shifts of two key loops (the entrance lid and the interface loop). Binding energy calculations demonstrate the enthalpy-entropy balance in the thermodynamic driving force of different inhibitors. The binding of inhibitors in monomeric form is entropy-driven, in which the solvation entropy from the binding-induced water restraints plays a key role, while the binding of inhibitors in dimeric form is enthalpy-driven, due to the promiscuous PSMA-inhibitor interactions. These insights into the molecular driving force of protein-ligand binding offer valuable guidance for rational drug design.