Activation of the epidermal growth factor receptor (EGFR) during tumor development can trigger the MEK signaling pathway. In the present study, we investigated the MEK signaling pathway in non‑small cell lung cancer (NSCLC) cells with respect to the effect of epidermal growth factor (EGF) on expression of Ret finger protein like 3 (RFPL3) and human telomerase reverse transcriptase (hTERT), and the effect of RFPL3 overexpression on other MEK signaling proteins. In vitro, A549 and H1299 cells were treated with different concentrations of EGF for 24 h or 48 h. Expression of RFPL3 and hTERT at the mRNA and protein levels was determined by real‑time quantitative PCR (RT‑qPCR) and western blot analysis; cell viability was detected by MTT assay, and apoptosis was assayed via flow cytometry. We also pretreated A549 and H1299 cells with EGFR tyrosine kinase inhibitors, AG1478 and erlotinib, and MEK‑specific inhibitor (PD98059) in the presence of EGF. We used western blot analysis to assess the expression levels of RFPL3, hTERT and related MEK‑pathway proteins in A549 and H1299 cells transfected with RFPL3‑overexpression plasmids. EGF significantly upregulated RFPL3 and hTERT protein levels in the NSCLC cells. RFPL3 and hTERT proteins upregulation by EGF were attenuated by pretreatment with AG1478 and erlotinib. EGF promoted proliferation and inhibited apoptosis; PD98059 decreased RFPL3 and hTERT protein expression; and RFPL3 overexpression increased the expression of hTERT and related MEK‑pathway proteins. EGF upregulated RFPL3 and hTERT protein expression in NSCLC cells via the MEK pathway, promoted cell proliferation and inhibited apoptosis. RFPL3 overexpression increased expression of hTERT and related MEK signaling proteins (Ras, Raf, ERK and p‑ERK), which implies that RFPL3 is a potential therapeutic target for NSCLC.