Cellular proteases were first known for their roles in processing peptides such as signal peptides. Recent discoveries have begun to unravel their multifunctional roles in many other biological processes including blood pressure regulation and virus infection. However, the knowledge gathered thus far is still limited regarding the underlying mechanisms for these functions. Therefore, my thesis research focuses on elucidating the structural and molecular basis for the multiple functions of several critical cellular proteases (aminopeptidase A (APA), dipeptidyl peptidase 4 (DPP4), proprotein convertases (PPC) and lysosomal cysteine proteases (LCP)). APA is well known for its role in blood pressure regulation, but the lack of its structure is a major obstacle for designing APA-targeting agents for the treatment of hypertension, which affects about one third of US adults. My study determined the crystal structures of APA in complex with a variety of substrates and inhibitors, and elaborated the substrate specificity of APA in physiological contexts. The structural information sheds light on the blood pressure regulation by APA, and lays the foundation for developing novel APA inhibitors to treat hypertension. DPP4 was recently identified as the receptor for the newly emerged Middle East respiratory syndrome coronavirus (MERS-CoV). To provide insights into the origin of MERS-CoV, I investigated the receptor usage and cell entry of MERS-CoV in comparison with HKU4, a related bat coronavirus. I discovered that DPP4 is also the receptor for HKU4. However, HKU4 failed to infect human cells because of its inability to use human cellular protease (e.g. PPC and LCP) for cell entry. PPC and LCP are critically involved in cell entry of many viruses. Here, I identified two residue differences between the spikes of MERS-CoV and HKU4 that account for their differential processes by human PPC and LCP, respectively. These results are critical for understanding the cross-species transmission of MERS-CoV, as well as for preventing the spread of related bat viruses. Taken together, the work presented in this thesis contributes to the overall understanding of the underlying mechanisms for diverse functions of cellular proteases in various health problems, and provides rationales for combating related health-threatening diseases.