The four and a half LIM domains 1 (FHL1) gene has been related to carcinogenesis. However, the expression status of FHL1 in human oral squamous cell carcinoma (OSCC) remains unclear and the detailed mechanism of gene silencing is poorly understood. The aim of this study was to examine the FHL1 expression level and its regulatory mechanism in OSCCs. Quantitative reverse-transcriptase-polymerase chain reaction (PCR) and western blotting showed significant downregulation of FHL1 in all OSCC-derived cell lines (Sa3, HSC-2, HSC-3, HSC-4, HO-1-u-1, HO-1-N-1, KON and Ca9-22) compared to human normal oral keratinocytes. We also found that FHL1 mRNA expression was frequently downregulated (P<0.01) in 51 (86.4%) of 59 primary OSCCs compared with the corresponding normal oral tissues, while there was no significant difference between the status of the FHL1 protein expression in OSCCs and the clinicopathological features. Using methylation-specific PCR, we detected methylated FHL1 in all cell lines and treatment with the DNA methyltransferase inhibitor, 5-aza-2'-deoxycytidine restored the FHL1 expression. However, no significant restoration of FHL1 expression was observed using sodium butyrate, an inhibitor of histone deacetylase and chromatin immunoprecipitation showed that histone H3 lysine 9 in the FHL1 promoter region was significantly acetylated. In addition, no mutation in the entire coding region of the FHL1 gene was found. Therefore, our data suggested that inactivation of the FHL1 gene is a frequent event during oral carcinogenesis and that the mechanism of FHL1 downregulation in OSCCs is through DNA methylation of the promoter region rather than histone deacetylation or mutation.