Biological porcine tissue was modified by the direct coupling of sulphonated poly(ethylene oxide) (PEO-SO3) containing amino acid end groups after glutaraldehyde fixation. The calcification of the modified tissue [bioprosthetic tissue (BT)-PEO-SO3] and control (BT control) was investigated by in vivo rate subdermal, canine aorta-illiac shunt and right ventricle-pulmonary artery shunt implantation models. Less calcium deposition of BT-PEO-SP3 than of BT control was observed in in vivo tests. Such a reduced calcification of BT-PEO-SO3 can be explained by decreases of residual glutaraldehyde groups, a space filling effect and, therefore, improved biostability and synergistic blood-compatible effects of PEO and SO3 groups after the covalent binding of PEO-SO3 to tissue. This simple method can be a useful anti-calcification treatment for implantable tissue valves.