Autophagy has recently attracted increasing attention for its role in conferring resistance to various commonly used anticancer therapies. Whereas its activities are known primarily to be under regulation of the high mobility group box-1 (HMGB1) gene, the expression of HMGB1 and its function in leukemia cells still remain unclear. In this study, we found that HMGB1 was expressed abundantly in various kinds of both leukemia and non-blood cancer cell-lines, and its expression was positively correlated with clinical status in childhood leukemia. In leukemia cells, when endogenous HMGB1 increased starvation-induced autophagy, this reaction was inhibited by the suppression of HMGB1. While the use of autophagy inhibitor, 3-methyladenine (3-MA), blocked the autophagic reaction and increased leukemia cell sensitivity to chemotherapy, enhancing HMGB1 expression decreased this sensitivity. Notably, suppressing HMGB1 expression also increased leukemia cell chemosensitivity. Furthermore, the phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin complex 1 (mTORC1) pathway was found to be functionally connected with HMGB1. HMGB1 gene transfection increased the LC3-II level and inhibited phosphorylation of Akt and p70S6K levels. Knockdown of HMGB1 expression blocked the association between mTOR and raptor in the setting of enhanced autophagy. When class I PI3K was inhibited by PI3K-I shRNA, it decreased the PI3K-I expression level. Knockdown of HMGB1 expression had no further effects on LC3-II. These results suggest that endogenous HMGB1 is an intrinsic regulator of autophagy in leukemia cells and it enhances leukemia cell chemoresistance likely through the PI3K/Akt/mTORC1 pathway.