The mechanisms underlying transthyretin-related amyloidosis in vivo remain unclear. The abundance of the 49-127 transthyretin fragment in ex vivo deposits suggests that a proteolytic cleavage has a crucial role in destabilizing the tetramer and releasing the highly amyloidogenic 49-127 truncated protomer. Here, we investigate the mechanism of cleavage and release of the 49-127 fragment from the prototypic S52P variant, and we show that the proteolysis/fibrillogenesis pathway is common to several amyloidogenic variants of transthyretin and requires the action of biomechanical forces provided by the shear stress of physiological fluid flow. Crucially, the non-amyloidogenic and protective T119M variant is neither cleaved nor generates fibrils under these conditions. We propose that a mechano-enzymatic mechanism mediates transthyretin amyloid fibrillogenesis in vivo. This may be particularly important in the heart where shear stress is greatest; indeed, the 49-127 transthyretin fragment is particularly abundant in cardiac amyloid. Finally, we show that existing transthyretin stabilizers, including tafamidis, inhibit proteolysis-mediated transthyretin fibrillogenesis with different efficiency in different variants; however, inhibition is complete only when both binding sites are occupied.