microRNAs (miRNAs) are small, non‑coding RNAs involved in multiple biological pathways by regulating post-transcriptional gene expression. Previously, autophagy has been reported to suppress the progression of non-small cell lung cancer (NSCLC). However, how miRNAs regulate autophagy in NSCLC remains to be elucidated. In the present study, the autophagy gene, autophagy-related 2B (ATG2B), was identified as a novel target of miR-143. The overexpression of miR-143 was able to downregulate the expression of atg2b at the transcriptional and translational levels by direct binding to its 3' untranslated region. Cell proliferation was significantly inhibited by the ectopic expression of miR-143 in H1299 cells. Knockdown of ATG2B resulted in a similar phenotype, with the overexpression of miR-143 in NSCLC cells. Furthermore, knockdown of ATG2B and hexokinase 2, a key enzyme in glycolysis and another target of miR-143, co-ordinated to inhibit the proliferation of H1299 cells. The results of the present study demonstrated that miR-143 was a novel and important regulator of autophagy by targeting ATG2B and repression of gene expression in autophagy and high glycolysis had a coordinate effect in H1299 cells. These results suggested that ATG2B may be a new potential therapeutic target for NSCLC. Furthermore, it was implied that interrupting autophagy and glycolysis improves NSCLC therapy.