We have developed a novel biodegradable matrix that has unusual cell adhesion and antithrombogenic properties. The prepared biodegradable matrix was alkali-treated collagen (AlCol) cross-linked with citric acid derivative (CAD), named as AlCol-CAD. The swelling ratio of AlCol-CAD decreased with increasing CAD concentration, but with a further increase of the CAD concentration, the swelling ratio of AlCol-CAD also began to increase; this behavior showed a point where the swelling ratio reached a minimum value before increasing. The highest shrinkage states of 7.5%, 15%, and 30% (w/v) in AlCol-CAD were observed at CAD concentrations of 10, 20, and 40 mM, respectively, and moreover, the residual amino groups in AlCol-CAD were found to decrease with increasing CAD concentration. On the other hand, increases in carboxyl groups of 7.5% and 15% (w/v) in AlCol-CAD were found at CAD concentrations higher than 10 and 20 mM, respectively, whereas, at CAD concentrations from 10 to 40 mM, no significant change of the carboxyl groups was observed in 30% (w/v) AlCol-CAD. Human umbilical vein endothelial cell (HUVEC) adhesion on 15% (w/v) AlCol-CAD increased with increasing CAD concentration up to 20 mM and then slightly decreased. In the case of 30% (w/v) AlCol-CAD, the number of adhered HUVECs on AlCol-CAD increased with increasing CAD concentration. Furthermore, it was observed that HUVECs had excellent cell proliferation on 15% (w/v) AlCol-CAD at CAD concentrations of 20 and 40 mM, after incubation for 7 days. No thrombus formation was observed on 15% (w/v) AlCol-CAD at CAD concentrations above 20 mM. These results suggested that the 15% (w/v) AlCol-CAD at CAD concentrations above 20 mM has both HUVEC adhesion and antithrombogenic properties.