Many studies have demonstrated that microRNA-210 (miR-210) expression is intensively upregulated in hypoxic states and differentially regulated in most types of cancer cells. However, the clinical significance of miR-210 and its effects on the response of leukemic cells to chemotherapeutic drugs in childhood acute lymphoblastic leukemia (ALL) remain unknown. In the current study, using real-time qRT-PCR to detect miR-210 expression in bone marrow samples from 114 children at initial diagnosis of ALL, we investigated the prognostic significance of miR-210 and determined its associations with common clinical characteristics and treatment outcome. We further examined its effect on the response to chemotherapeutic drugs in the Reh and RS4;11 cell lines. Results showed that miR-210 expression was significantly lower in patients suffering from relapse and induction failure than in other patients (P < 0.001). Using the receiver operating characteristic curve, 3.8243 was selected as the cut-off value of miR-210 expression in our test cohort (38 cases). A significantly poorer treatment outcome (P < 0.05) was found in the low-expression group and verified in the validation cohort (76 cases, P < 0.05). Patients with low expression of miR-210 and positive minimal residual disease at the end of induction had a much higher rate of relapse or induction failure (P = 0.001). Increasing/decreasing miR-210 expression using agomir/antagomir could enhance or reduce the response of Reh cells and RS4;11 cells to daunorubicin/dexamethasone/L-asparaginase and daunorubicin/dexamethasone/vincristine, respectively. In conclusion, miR-210 may be a good prognostic factor and a useful predictor of drug sensitivity, and is a potential therapeutic target for pediatric ALL.