The present study aimed to investigate the expression, biological function and mechanism of action of engrailed homeobox 2 (EN2) in non-small cell lung cancer (NSCLC) at the tissue and cellular level. A total of 42 patients who underwent surgical resection of NSCLC tissues between January 2014 and January 2015 were included in the present study. EN2 mRNA expression levels in explanted NSCLC tissues were determined using reverse-transcription quantitative polymerase chain reaction analysis. Adenocarcinoma human alveolar basal epithelial A549 cells were transfected with negative control plasmids or those containing EN2, enabling its overexpression. To assess the effect of EN2 overexpression in A549 cells, a Cell Counting kit-8 assay was used to analyze cellular proliferation, a Transwell assay was used to evaluate cellular migration and invasion and flow cytometry was used to detect the cell cycle distribution. To measure protein expression of EN2 and β-catenin in A549 cells, western blotting was also conducted. EN2 mRNA expression levels in NSCLC tissues were lower than those in normal tissues, and were associated with metastasis, clinical staging and differentiation degrees of NSCLC. Increased expression of EN2 inhibited the proliferation of A549 cells , and suppressed their migration and invasion. Elevated EN2 expression inhibited the proliferation of A549 cells by regulating the G/S phase transition. β-catenin protein expression levels and nuclear translocation in A549 cells were inhibited by EN2 overexpression. The present study demonstrated that expression of EN2 in NSCLC tissues was downregulated and negatively associated with the degree of disease differentiation, lymphatic metastasis and clinical staging. Overexpression of EN2 inhibits the proliferation, migration and invasion of A549 cells, as well as the expression of β-Catenin and nuclear translocation.