We analyzed the mutational and methylation status of the spleen tyrosine kinase (Syk) gene and both mRNA and protein levels in primary oral squamous cell carcinoma (OSCC) and OSCC-derived cell lines and examined the function of the Syk gene in OSCC-derived cell lines in vitro. Using quantitative real-time reverse transcription polymerase chain reaction, Western blotting and immunofluorescence on 7 OSCC-derived cell lines and normal oral keratinocytes (NOKs), Syk mRNA and protein expression were commonly downregulated in all cell lines compared to the NOKs. Although no sequence variation in the coding region of the Syk gene was identified in these cell lines, we found frequent hypermethylation in the CpG island region. Syk expression was restored by experimental demethylation. In addition, using a wound healing assay and in vitro invasion assay, we performed functional analysis using Syk transfected into the OSCC-derived cell lines, and they showed significant inhibition of motility and invasiveness. In clinical samples, high frequencies of Syk downregulation were detected by immunohistochemistry (33 of 53 [62%]). Furthermore, the Syk expression status was correlated significantly (p = 0.047) with tumor metastasis to cervical lymph nodes. These results suggest that the Syk gene is frequently inactivated during oral carcinogenesis and that an epigenetic mechanism may regulate loss of expression possibly leading to metastasis.