Oestrogen receptor beta (ERβ) and epidermal growth factor receptor (EGFR) pathway can synergistically promote the proliferation, invasion, and metastasis of non-small-cell lung cancer (NSCLC) cells. ERβ has five subtypes, and the selective splicing of exon 8 in ERβ5 transcription translational phase makes its biological function different from other subtypes. The following study investigates whether ERβ5 interacts with EGFR pathway in lung cancer. Briefly, we found that the overexpression of ERβ5 and EGFR is associated with poor prognosis and decreased overall survival in NSCLC patients. Furthermore, the effects of ERβ5 and EGFR on cell biological behaviour were investigated in vitro. These results indicated that the combination of ERβ5 and EGF induces cell proliferation and invasion, while the combination of ERβ5 and Gefitinib (EGFR inhibitors, Gef) induces cell apoptosis and promotes cell mitosis in A549 cell line. In addition, the combination of ERβ5 and EGF increases the expression of ERβ5, EGFR, and p-ERK1/2 in lung cancer cells. To sum up, the obtained results suggest that ERβ5 and EGFR synergistically promote the progression of lung cancer by activating MEK/ERK signalling pathway, which provides a theoretical basis for more accurate combined targeted therapy.