The urinary bladder must remain distensible enough to expand as urine is produced yet strong enough to expel urine without rupture. Disruption in the arrangement of the bladder wall extracellular matrix (ECM) can lead to alterations in mechanical compliance, disrupting normal function and resulting in bladder dysfunction. Previously, we discovered that the mast cell activator compound 48/80 increased detrusor excitability and bladder wall mechanical compliance, resulting in increased urination frequency in mice in a mast cell-independent manner. While the effects on detrusor excitability depended on urothelial prostaglandin production, the mechanisms by which compound 48/80 altered mechanical compliance remained unclear. This study tests the hypothesis that compound 48/80 increases compliance through activation of matrix metalloproteases (MMPs) in the bladder wall. Using the Pentaplanar Reflected Image Macroscopy (PRIM) System and multiphoton microscopy, we measured ex vivo intravesical pressure, mechanical compliance, wall thickness, and permeability in bladders from C57Bl/6 mice before and after exposure to compound 48/80. Intravesical instillation of compound 48/80 increased mechanical compliance and reduced wall thickness without affecting urothelial permeability. This change in compliance was inhibited by the nonspecific MMP inhibitor doxycycline and the MMP-2 inhibitor ARP100. Bladder MMP-2 activity increased, and TIMP-2 activity decreased, after incubation with compound 48/80 as measured by zymography. Together, these findings suggest that compound 48/80 changes bladder mechanical compliance through ECM breakdown caused by rapid activation of MMP-2 and inhibition of TIMP-2. Moreover, these effects occur without altering urothelial permeability, suggesting a novel receptor-mediated mechanism and not a direct effect of compound 48/80 on MMP-2 or TIMP-2.