A carboxylated pullulan, for use as a structural material for a number of tissue engineering applications, was synthesized and conjugated with heparin. By immobilization of heparin to pullulan, endothelial cells (ECs) attached on the heparin-conjugated pullulan were more aggregated than when attached to other pullulan derivatives. Attachments were 50, 45, 49, and 90% for a polystyrene dish, pullulan acetate, carboxylated pullulan, and heparin-conjugated pullulan, respectively. Heparin-conjugated pullulan inhibited the proliferation of smooth muscle cells (SMCs) in vitro. Heparin-conjugated pullulan material can thus be used for the proliferation of vascular ECs and to inhibit the proliferation of SMCs.