MicroRNAs (miRNAs) function as post-transcriptional mediators for genes involved in cancer progression, including chronic lymphocytic leukemia. MiR-582-5p has been identified as a tumor suppressor in various tumors. The antioncogenic role of miR-582-5p was then validated in this study. Mononuclear cells were isolated from peripheral blood samples of patients with chronic lymphocytic leukemia and healthy donors. Expression of miR-582-3p in the mononuclear cells was examined by qRT-PCR. CCK8 assay was performed to detect cell viability, and cell cycle and apoptosis were evaluated by flow cytometry. Dual luciferase activity assay was performed to determine the targeting relationship between miR-582-3p and HNRNPA1, and western blot was performed to unravel the mechanism. MiR-582-5p was reduced in mononuclear cells of patients with chronic lymphocytic leukemia compared to healthy donors. Forced miR-582-5p expression reduced cell viability, and promoted apoptosis of chronic lymphocytic leukemia cells. Cell cycle was arrested in G0/G1 phase via miR-582-5p mimic. MiR-582-5p bound to HNRNPA1 (heterogeneous nuclear ribonucleoprotein A1) and down-regulated its expression. Silence of HNRNPA1 decreased cell viability, promoted apoptosis, and blocked cell cycle at G0/G1 phase through up-regulation of IκBα (IkappaBalpha). Moreover, HNRNPA1 silencing attenuated the promotive effect induced by miR-582-5p inhibitor on the progression of chronic lymphocytic leukemia. MiR-582-5p demonstrated anti-proliferative and pro-apoptotic roles in chronic lymphocytic leukemia cell growth via down-regulation of HNRNPA1 and up-regulation of IκBα, thus inactivating NF-κB.