Abstract: Objective To explore the influence of Clostridium butyricum on the expression of vascular endothelial cell growth factor (VEGF) and its receptor 2 (VEGFR-2), proliferating cell nuclear antigen (PCNA), and tight junction protein claudin-2 in intestinal tissue in newborn rat with necrotizing enterocolitis (NEC). Methods Forty-eight-hour-old Sprague-Dewley (SD) rats were randomly divided into model group, control group, low-dose group, mid-dose group, and high-dose group, 12 rats each. Rats in each group were fed with milk substitute. The NEC model were created by hypoxia and cold stimulation for 3 consecutive days in model group, low-dose group, mid-dose group, and high-dose group. Meanwhile, low-dose group, mid-dose group, and high-dose group were intervened by being fed with Clostridium butyricum 0.2, 0.4, and 0.8 g/(kg·d), respectively. All rats in each group were sacrificed  on day 4 and the intestines tissue was obtained. The pathological changes had been observed. The expression of VEGF, PCNA, and claudin-2 were detected by immunohistochemistry. The expression of VEGFR-2 was detected by RT-PCR. Results The intestines pathological scores was significantly different among five groups (P < 0.05) with the scores being higher in model group than in lowdose group, mid-dose group, high-dose, group and control group, and being higher in all intervention groups than in control group (P all < 0.05). The expressions of VEGF, VEGFR-2, and claudin-2 in model group were higher than those in all intervention groups and control group with them being higher in all intervention group than in control group (P all < 0.05). The expression of PCNA in model group was lower than those in all intervention groups and control group with it being lower in all intervention groups than that in control group (P all < 0.05). The expression of VEGF, VEGF-2, PCNA, and claudin-2 were no different among intervention groups (P all > 0.05). Conclusion The expression of VEGF, VEGF-2, and claudin-2 were higher in rats with NEC, while the expression of PCNA was lower. Supplementation of Clostridium butyricum may protect newborn rats by its act on these factors.