It was suggested in our previous studies that carbodiimide-and genipin-crosslinked gelatin hydrogels could be used as bioadhesives to overcome the cytotoxicity problem associated with formaldehyde-crosslinked gelatin hydrogels. In this study, we investigated the crosslinking structures of carbodiimide-and genipin-crosslinked gelatin hydrogels. We found that crosslinking gelatin hydrogels with carbodiimide or genipin could produce distinct crosslinking structures because of the differences in their crosslinking types. Carbodiimide could form intramolecular crosslinks within a gelatin molecule or short-range intermolecular crosslinks between two adjacent gelatin molecules. On the basis of gel permeation chromatography, we found that the polymerization of genipin molecules could occur under the conditions used in crosslinking gelatin hydrogels via a possible aldol condensation. Therefore, besides intramolecular and short-range intermolecular crosslinks, additional long-range intermolecular crosslinks could be introduced into genipin-crosslinked gelatin hydrogels. Crosslinking a gelatin hydrogel with carbodiimide was more rapid than crosslinking with genipin. Therefore, the gelation time for the carbodiimide-crosslinked gelatin hydrogels was significantly shorter than that of the genipin-crosslinked gelatin hydrogels. However, the cohesive (interconnected) structure of the carbodiimide-crosslinked gelatin hydrogels was readily broken because, unlike the genipin-crosslinked gelatin hydrogels, there were simply intramolecular and shortrange intermolecular crosslinks present in the carbodiimidecrosslinked hydrogel. In the cytotoxicity study, the carbodiimide-crosslinked gelatin hydrogels were dissolved into small fragments in the cultural medium within 10 min. In contrast, the genipin-crosslinked gelatin hydrogels remained intact in the medium throughout the entire course of the study. Again, this may be attributed to the differences in their crosslinking structures. The genipin-crosslinked gelatin hydrogels were less cytotoxic than the carbodiimidecrosslinked gelatin hydrogels.