Gaining insight into the in situ receptor-ligand binding is pivotal for revealing the molecular mechanisms underlying the physiological and pathological processes and will contribute to drug discovery and biomedical application. An important issue involved is how the receptor-ligand binding responds to mechanical stimuli. This review aims to provide an overview of the current understanding of the effect of several representative mechanical factors, such as tension, shear stress, stretch, compression, and substrate stiffness on receptor-ligand binding, wherein the biomedical implications are focused. In addition, we highlight the importance of synergistic development of experimental and computational methods for fully understanding the in situ receptor-ligand binding, and further studies should focus on the coupling effects of these mechanical factors.