Evaluation of cathepsin B activities for degrading collagen IV and heat-denatured collagen IV (gelatin) were performed by surface plasmon resonance (SPR) and circular dichroism (CD) measurements. The optimal pH of cathepsin B activity for degrading each substrate was around 4.0. The ΔRU(15 min), which is a decrease in the SPR signal at 15 min after injection of cathepsin B, was smaller for collagen IV than for heat-denatured collagen IV owing to the presence of triple-helical conformation. An unstable nature of the triple-helical conformation of collagen IV at pH 4.0 was shown by the CD study. Degrading collagen IV by cathepsin B was facilitated owing to a local unwinding of the triple-helical conformation caused by proteolytic cleavage of the non-helical region. The concentration dependence of the initial velocity for degrading collagen IV by cathepsin B at pH 4.0 was biphasic, showing that cathepsin B at low concentration exhibits exopeptidase activity, while the enzyme at high concentration exhibits endopeptidase activity. The kinetic parameters for the exopeptidase activity of cathepsin B toward collagen IV and heat-treated collagen IV were evaluated and discussed in terms of the protease mechanism.