It is well known that the Homo sapiens LIM homeobox domain 6 gene (hLHX6), a putative transcription regulator, controls the differentiation and development of neural and lymphoid cells, particularly in the central nervous system. In this study, we investigated hLHX6.1 (an isoform of hLHX6), which functions as a tumor suppressor gene in the cervix. Firstly, the methylation levels of the hLHX6 and hLHX6.1 promoters were investigated in 8 cervical cancer cell lines and human tissue samples with a distinctive degree of malignant transformation. In spite of the presence of multiple cytosine guanine dinucleotides (CpG islands) in 2 proximal promoters of the hLHX6 and hLHX6.1 genes, only the hLHX6.1 promoters were found to be mostly hypermethylated and associated with transcriptional silencing by promoter methylation, whereas the hLHX6 promoters were not. Methylation levels in the hLHX6.1 promoter were also found to be strongly related to cervical cancer development. The level of hLHX6.1 gene expression was found to be relatively high in normal cells, in which the hLHX6.1 promoter was mostly unmethylated. However, the hLHX6.1 gene expression was down-regulated or undetectable in cervical cancer cell lines and cancer tissues, in which the hLHX6.1 promoter was hypermethylated. This epigenetic alteration in the hLHX6.1 promoter begins at a relatively early stage, suggesting its potential as a biomarker for the early diagnosis and prevention of cervical cancer. Moreover, the overexpression of the hLHX6.1 gene in cervical cancer cells suppressed the tumorigenic phenotype, as shown by soft agar colony formation and migration assays, suggesting that hLHX6.1 could be a new tumor suppressor gene in the cervix.