Hesperetin, a predominant flavonoid found in citrus fruits, has received considerable attention for its potential anticancer activity through the reduction of cell viability and the induction of apoptosis. Several effector mechanisms have been demonstrated underlying the antitumor properties of hesperetin but its specific mechanisms have not yet been fully elucidated. In the present study, how hesperetin affects the proliferation of A549 cells and the related cell proliferation regulatory mechanisms, were inevstigated. To elucidate the mechanisms underlying the effects of hesperetin on A549 cells, MTT assay, colony formation assay, flow cytometry, immunoblotting, reverse transcription­quantitative PCR and JC­1 staining were performed. The data revealed that hesperetin inhibited cell proliferation and induced apoptosis in these cells. Hesperetin also decreased the level of heat shock protein 70 (Hsp70), a negative regulator of the mitochondrial apoptosis pathway, often overexpressed in various cancer cells and suspected to contribute to tumor development. Hesperetin­induced Hsp70 suppression was associated with reduced cytosolic Bax and increased mitochondrial Bax levels, leading to the enhancement of the mitochondrial apoptotic cascade. The Hsp70 overexpression­induced reduction in the level of hesperetin­induced apoptosis provides evidence to hesperetin­induced apoptosis being mediated by affecting Hsp70. Furthermore, it was demonstrated that hesperetin reduced Hsp70 expression by inducing a proteasome­mediated degradation via the upregulation of E3­ligase, C­terminus of Hsp70­interacting protein (CHIP). The present study highlighted the importance of the Bax activation­triggered mitochondria­mediated pathway for hesperetin­induced apoptosis and demonstrated a novel mechanism of how Hsp70 played a critical role in the negative regulation of this apoptotic network in cancer cells.