MicroRNA (miR)-7 has been reported to act as a suppressor in several types of cancer, including non-small cell lung cancer (NSCLC). In addition, paired box 6 (Pax6), a highly conserved transcriptional factor, has been implicated in NSCLC. However, the exact role of miR-7, and the association between miR-7 and Pax6 in NSCLC cells remain to be fully elucidated. The present study demonstrated that miR-7 was downregulated and Pax6 was upregulated in NSCLC cell lines. Subsequently, it was demonstrated that overexpression of miR-7 notably inhibited the protein expression of Pax6, while inhibition of miR-7 enhanced the protein expression of Pax6 in NSCLC A549 cells. Further investigation identified Pax6 as a target of miR-7 in A549 NSCLC cells. Ina ddition, the overexpression of miR-7 significantly inhibited A549 cell proliferation and invasion, which was reversed by upregulation of Pax6. Investigation of the underlying molecular mechanism revealed that the extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase (MAPK) signaling pathways were downregulated in the miR-7-overexpressed A549 cells, but were activated in the Pax6-overexpressed A549 cells. Based on these findings, it was suggested that miR-7 negatively regulates the protein level of Pax6, which can promote the proliferation and invasion of NSCLC cells via activation of the ERK and MAPK signaling pathways. Therefore, miR-7/Pax6 may offer potential for use as a target for the treatment of NSCLC.