Desmosomes are intercellular junctions that confer strong cell-cell adhesion thus providing resistance against mechanical stress on epithelial tissues. A body of evidence indicates that decreased expression of desmosomal proteins is associated with poor prognosis in various cancers. As a key component of desmosomal plaque proteins, the functional role of desmoplakin (DSP) in cancer is not yet elucidated. Here, we reported the anti-tumourigenic activity of DSP in non-small cell lung cancer (NSCLC). We found that DSP expression was downregulated in 8 out of 11 lung cancer cell lines and in 34 out of 56 primary lung tumours by DSP DNA methylation. Ectopic expression of DSP in the NSCLC cell line H157 significantly inhibited cell proliferation, anchorage-independent growth, migration, as well as invasion, and also increased the sensitivity of NSCLC cells to apoptosis induced by an anticancer drug, gemcitabine. Furthermore, overexpression of DSP enhanced expression of plakoglobin (γ-catenin), resulting in decreased TCF/LEF-dependent transcriptional activity and reduced expression of the Wnt/β-catenin target genes Axin2 and MMP14. In accordance, DSP suppression by siRNA resulted in downregulation of plakoglobin and upregulation of β-catenin and MMP14. Taken together, these data suggest that DSP is inactivated in lung cancer by epigenetic mechanism, DSP increases the sensitivity to anticancer drug-induced apoptosis, and DSP has tumour suppressive function possibly through inhibition of the Wnt/β-catenin signalling pathway in NSCLC cells. The epigenetic regulation of DSP and its ability to increase the sensitivity to anticancer drug-induced apoptosis has potential implications for clinical application.