LIM homeobox domain 6 (LHX6) is a putative transcriptional regulator that controls the differentiation and development of neural and lymphoid cells. However, the function of LHX6 in cancer development remains largely unclear. Recently, we found that LHX6 is hypermethylated in lung cancer. In this study, we analysed its epigenetic regulation, biological functions, and related molecular mechanisms in lung cancer. Methylation status was evaluated by methylation-specific PCR and bisulfite genomic sequencing. LHX6 mRNA levels were measured in relation to the methylation status. The effects of LHX6 expression on tumourigenesis were studied in vitro and in vivo. LHX6 was readily expressed in normal lung tissues without methylation, but was downregulated or silenced in lung cancer cell lines and tissues with hypermethylation status. Treatment of lung cancer cells with the demethylating agent 5-aza-2'-deoxycytidine restored LHX6 expression. Moreover, LHX6 hypermethylation was detected in 56% (52/93) of primary lung cancers compared with none (0/20) of the tested normal lung tissues. In lung cancer cell lines 95D and H358, forced expression of LHX6 suppressed cell viability, colony formation, and migration, induced apoptosis and G1/S arrest, and inhibited their tumorigenicity in nude mice. On the other hand, knockdown of LHX6 expression by RNA interference increased cell proliferation and inhibited apoptosis and cell cycle arrest. These effects were associated with upregulation of p21 and p53, and downregulation of Bcl-2, cyclinD1, c-myc, CD44, and MMP7. In conclusion, our results suggest that LHX6 is a putative tumour suppressor gene with epigenetic silencing in lung cancer.