Background and Aim: The incidence of acute lymphoblastic leukemia (ALL) is highest in childhood malignant tumor in China. The high-dose methotrexate (HDMTX) treatment is very effective in ALL, and it can improve event-free survival rate. However, while executing the anti-tumor effect, it produces highly toxic effects on rapidly dividing cells which are normal. It seems probable that the HDMTX treatment injures intestinal mucosal barrier. The changes of intestinal mucosal barrier can be evaluated through measuring the level of plasma endotoxin and diamine oxidase (DAO). Method: Blood samples were collected from 30 normal children and 30 children with ALL at 1h, 24h, 44h and 68h after HDMTX. The levels of plasma endotoxin and DAO were measured at 1h, 24h, 44h and 68h after HDMTX with spectrophotometry. The levels of endotoxin and DAO were also measured in 4 different courses in 7 children with ALL. Results: The levels of plasma endotoxin and DAO at 1h, 24h, 44h and 68h after HDMTX were higher than in normal children (P<0.01). The levels of plasma endotoxin and DAO at 24h and 44h after HDMTX were both higher than at 1h and 68h (P<0.01). There was no significant difference found in the measured results of plasma endotoxin and DAO at 1h and 68h after HDMTX (P>0.05). There was no significant difference found in the increased levels of endotoxin and DAO at 1h, 24h, 44h and 68h after HDMTX in 4 different courses of 7 children with ALL(P>0.05). Conclusion: By measuring the level of plasma endotoxin and DAO in children with ALL and during HDMTX chemotherapy, the results suggest that there is increased intestinal permeability.