Bisphosphonates (BPs) are the drug of choice for treating bone diseases such as osteoporosis, Paget's disease, and metastatic bone disease. BPs with nitrogen-containing side chains (N-BPs) are known to act as inhibitors for farnesyl pyrophosphate synthase (FPPS), a key enzyme in the mevalonate pathway. In this study, we evaluated the effect of different side chains on the binding affinity of BPs to human FPPS using calorimetric techniques. Differential scanning calorimetry (DSC) was used to determine the thermal unfolding of FPPS in the presence of BPs. The addition of a series of clinically available BPs increased the structural stability of human FPPS by preferential binding, as indicated by an increase in the FPPS unfolding temperature. The magnitude of the increase was correlated with in vivo antiresorptive efficacy, suggesting that the stabilization of FPPS underlies the inhibitory effect of the BPs. Isothermal titration calorimetry (ITC) experiments were performed to evaluate the binding thermodynamics of BPs against human FPPS. Analysis of the binding energetics revealed that over 30 years of optimization practiced by different pharmaceutical companies has enhanced the enthalpic contribution as well as binding affinity of BPs. The larger enthalpic contribution observed for newer, more potent BPs derives from both improved hydrogen bonding interactions and shape complementarity based on comparisons of our results with available structure information.