ObjectiveTo detect 5-FU concentration and investigate the changes of pathology， and Ki-67 protein expression after intraoperative regional chemotherapy (RC) for colon cancer. MethodsAll the patients were randomized into two groups: RC group (n=20), received intraoperational RC with 100 ml physiological saline contained 5-FU (15 mg/kg) and camptothecine (0.06 mg/kg)； control group (n=20), saline alone. The samples from portal vein blood, peripheral blood, peritoneal fluid， and peri-cancerous tissues in RC group were taken to detect the 5-FU concentration by high performance liquid chromatography (HPLC), respectively at 2, 5, 10, 20, 30, and 60 minutes after treatment. The pathological changes were observed and Ki-67 protein expressions were examined by immunohistochemical staining for all the cancer tissues postoperatively in two groups. ResultsPeak concentration of 5-FU appeared at 2 min after treatment, and decreased gradually. 5-FU concentration in peritoneal fluid was the highest, and the lowest in the peripheral blood (Plt;0.01). In RC group， light karyopyknosis, nuclear swelling, and coagulative necrosis of cancer cells, and light intercellular substance hydropsia, inflammatory cells invasion were observed under light microscopic examination; light vasculitis presented also in five cases. Nuclear swelling, heterochromatin agglutination, perinuclear gap expansion, mitochondrial swelling, endoplasmic reticulum expansion, and Golgi complex expansion were observed with transmission electron microscope. Ki-67 protein expression of colon cance tissues in RC group was lower than that in control group （Plt;0.05）. Conclusions Intraoperative RC for colon cancer may sustain a high concentration of chemotherapy drugs in peritoneal fluid and portal vein blood, and alter histopathological morphology of cancer cells, and suppress Ki-67 protein expression. So, intraoperative RC may play an important role in preventing intraoperative spreading and postoperative recurrence of colon cancer.