Objective To observe the histopathological changes of tumor tissue after intraoperative regional chemotherapy of rectal cancer. Methods After the treatment of intraoperative regional chemotherapy with 5-FU (15 mg/kg)， the histopathological changes of tumor tissue were detected. Results Slight changes with cancer cells in all the cases accepted intraoperative regional chemotherapy were found under light microscope such as karyopyknosis, nuclear swelling, coagulation and necrosis of cytoplasm, hydropsia of intercellular substance, invasion of inflammatory cells, and 9/15 cases with slight inflammation of vessels were observed; While those changes were found in individual cells of the cases without regional chemotherapy. The more enlarged intercellular space of cancer cell was observed under electron microscope in the case with regional chemotherapy. Conclusion The intraoperative regional chemotherapy of rectal tumor can change the histopathological appearance of tumor tissue, that is significant in preventing cancer cells diffusing during operation and relapsing after operation.