Objective:The mechanism responsible for the regulation of HSPB1 expression in oral squamous cell carcinoma was explored in this study. Study Design: The expression and the methylation status of HSPB1 in oral squamous carcinoma cells were examined using real-time reverse transcription-PCR, methylation-specific PCR and pyrosequencing. Results: HSPB1 expression was weakly expressed in oral squamous carcinoma cell lines (N = 4) as compared to that of normal human oral keratinocytes. The lower expressed HSPB1 was associated with prmoter hypermethylation of the HSPB1 gene, and the expression of HSPB1 could be induced by treating the cells with a DNA methyltransferase inhibitor, RG108. Promoter hypermethylation of the HSPB1 gene was also noted in primary oral squamous carcinomas, concomitant with reduced levels of HSPB1 gene expression, as compared to those of the paired neighboring normal tissues. Conclusion: Aberrant promoter hypermethylation of the HSPB1 gene may explain the reduced expression of HSPB1 noted in oral cancer cells.