The homo sapiens LIM homeobox domain LHX6 gene, hLHX6, is a putative transcription regulator with homeo-domain. Multiple cytosine guanine dinucleotides (CpG island) are found in the genomic sequences between exon 4a and exon 5 of the gene encoding hLHX6s (alternative short iso-form of hLHX6 gene). This specific CpG island, hLHX6-HMR, is found frequently hypermethylated in 7 cervical cancer cell lines as shown in MSP, BSP, and COBRA assays. Methylation densities were also investigated with human tissue samples with a distinctive degree of malignant transformation. Our data showed that the hLHX6-HMR was rarely or partly methylated in the normal and CIN I cells, respectively. In contrast, it was frequently hypermethylated in CIN II, CIN III, and invasive carcinoma cells. In summary, this methylation study led to two conclusions. First, hLHX6-HMR hypermethylation is exclusively associated with cervical carcinogenesis. Second, the epigenetic change in hLHX6-HMR seems to start at CIN I, relatively early stage of cervical cancer development. Therefore, hLHX6-HMR can be used as an effective and sensitive methylation biomarker for early diagnosis of cervical cancer.