Abstract <p><b>Objective:</b> To evaluate the effect of enzymatic collagen degradation on the subsequent calcification of epoxy-treated bovine pericardium used in the manufacture of bioprosthetic heart valves in an in vitro experiment. <b>Methods:</b> The study material consisted of patches of epoxy-treated bovine pericardium. Fragments of this material were incubated in a solution of bacterial collagenase. The degree of sample degradation following enzyme treatment was analyzed based on mass loss and changes in mechanical properties. Samples that underwent proteolysis were also placed in a calcifying solution for 3 weeks, after which their calcium content was determined using a spectrophotometric method. The control in the experiments were fragments of epoxy-treated pericardium that were not exposed to collagenase. <b>Results:</b> On average, the mass loss of epoxy-treated pericardium fragments after 24-h incubation in collagenase was about 8%, which is comparable to values reported in the literature for glutaraldehyde-fixed pericardium. Enzyme treatment also led to a reduction in the ultimate tensile strength of the studied material (14.15 [13.08–16.58; 11.4–22.43] MPa (median [25th–75th percentile; min–max]) in the control group versus 10.67 [7.37–11.6; 6.39–11.95] MPa in the experimental group, <i>p</i> = 0.0003) and enhanced its calcification by a factor of 1.6 (8.61 [6.58–9.81; 4.78–14.53] mg calcium/g tissue in the control group versus 13.41 [10.58–17.27; 7.76–28.41] mg calcium/g tissue in the experimental group, <i>p</i> = 0.0001). <b>Conclusion:</b> Enzymatic collagen degradation in epoxy-treated bovine pericardium enhances its calcification in vitro. The obtained data suggest that the accumulation of collagenolytic enzymes in the leaflets of xenopericardial bioprosthetic heart valves is an important mechanism of structural valve degeneration.</p>