Abstract: Objective To evaluate the value of fecal high mobility group box-1 (HMGB1) in early diagnosis and prediction of the severity of neonatal necrotizing enterocolitis (NEC). Methods From July 2013 to June 2015, the neonates who had distention, vomit, or gross blood in stool and were suspected of NEC were recruited as NEC group while hospitalized children without abdominal distension, vomiting, bloody diarrhea, or other gastrointestinal symptoms were recruited as the control group. Stool samples were collected on day 1, 3, 5 and 7 after admission. The level of HMGB1 was measured by enzyme linked immunosorbent assay (ELISA). Results In the end, there were 46 cases in NEC group and 15 cases in control group. In NEC group, 29 cases were confirmed of stage Ⅰ of NEC by abdominal radiograph within 24 h after hospitalized, all of them were deteriorated to stage Ⅱ phase in 4 days, and 10 cases were deteriorated to stage Ⅲ . Seventeen cases were confirmed of stage Ⅱ by abdominal radiograph within 24 h after hospitalized, 7 cases were deteriorated to stage Ⅲ. In 17 stage Ⅲ cases, 11 cases received surgical treatment and 6 cases gave up. Eight cases survived and 3 died after surgery. The levels of HMGB1 in NEC group on day 1, 3, 5 and 7 after hospitalized were higher than those in control group (P < 0.05). With the exacerbation of NEC from stage I to Ⅲ , the levels of HMGB1 increased gradually (P < 0.05). Conclusions With the exacerbation of NEC, the level of HMGB1 is gradually increased, which indicates that it has certain clinical value in early diagnosis and prediction of severity of NEC.