Acute lymphoblastic leukemia (ALL) is characterized by abnormal lymphoblasts accumulation in the bone marrow and blood. Despite great efforts have been made in exploring novel therapeutic method, the prognosis of children with ALL is still unsatisfied. Glucocorticoid (GC) resistance is a great obstacle for the clinical treatment of ALL. Therefore, it is essential to investigate the molecular mechanism underlying the GC resistance. According to previous reports, long noncoding RNAs (lncRNAs) are involved in drug resistance of various human cancers. LncRNA HOXA cluster antisense RNA2 (HOXA-AS2) has been reported in several human malignancies due to its oncogenic property. However, the molecular mechanism of HOXA-AS2 involved in the GC resistance of ALL still needs to be further clarified. At first, we found that lncRNA HOXA-AS2 was highly expressed both in prednisone insensitive ALL cell lines and patient samples. Gain or loss-of-function assays revealed that HOXA-AS2 enhanced GC resistance via promoting cell proliferation and inhibiting cell apoptosis. Furthermore, we validated that HOXA-AS2 upregulated HOXA3, thereby activating EGFR/Ras/Raf/MEK/ERK signaling pathway. Our findings showed that HOXA-AS2 may be a potential therapeutic target for ALL patients with poor GC resistance.