A previous study demonstrated that ethylenediamine-conjugated catalase (ED-catalase) inhibits peritoneal dissemination of tumor cells in mice. To increase its inhibitory effects by sustained release, a hydrogel formulation of ED-catalase was prepared using a biodegradable hydrogel consisting of an acidic gelatin with an isoelectric point of 5.0. Although intraperitoneally injected ED-catalase solution rapidly disappeared from the cavity, more than 10% of ED-catalase remained even at 14 days after implantation of ED-catalase/hydrogel into the cavity. Then, the effect of ED-catalase/hydrogel on peritoneal dissemination of tumor cells was evaluated by measuring the luciferase activity of abdominal organs after intraperitoneal inoculation of colon26/Luc, a colon adenocarcinoma stably expressing luciferase. ED-catalase/hydrogel showed a significantly (P<0.05) greater effect on inhibiting the growth of tumor cells than ED-catalase solution, demonstrating the importance of the retention of ED-catalase within the cavity as far as inhibition is concerned. Serial in vivo images of luciferase activity revealed that the ED-catalase/hydrogel significantly (P<0.05) retarded the growth rate of tumor cells. Survival of tumor-bearing mice supported the findings obtained with the luminescence-based analyses. These findings indicate that the sustained release of ED-catalase from hydrogels into the cavity is highly effective in inhibiting the growth of peritoneally disseminated tumor cells.