Homeobox genes comprise a family of regulatory genes that contain a common homeobox domain and act as transcription factors. Changes in the expression of homeobox genes are observed in many cancers, including leukaemia, colon, skin, prostate, breast, and ovarian cancers. Recent studies indicate that homeobox A5 (HOXA5) may serve as a tumour suppressor gene in breast cells. However, the precise role of HOXA5 in lung cancer remains unclear. Using microarrays and an invasion/metastasis lung adenocarcinoma cell line model, we showed that the expression of HOXA5 is negatively correlated with cancer cell invasion ability. The ectopic expression of HOXA5 in highly invasive cancer cells suppressed cell migration, invasion, and filopodia formation in vitro and inhibited metastatic potential in vivo. The knockdown of HOXA5 expression via a HOXA5-specific siRNA was able to promote the invasiveness of lung cancer cells. Furthermore, HOXA5 expression was associated with improved overall survival and disease-free survival in non-small cell lung cancer patients with wild-type EGFR (P = 0.0199 and 0.0345, respectively). Genome-wide transcriptome and pathway analyses indicated that these effects of HOXA5 may be associated with the regulation of cytoskeletal remodelling. In summary, our studies provide new insights into how HOXA5 may contribute to the suppression of metastasis in lung cancer.