Lymph node metastasis is the hallmark of malignant neoplasms in patients of oral cancer, accounting for the poor diagnosis and reduced 5-year survival rate. Here we sought to identify cell motility-associated proteins of oral cancer by proteomic approach. We compared the proteomes of two oral cancer cells, CAL-27 and SAS, with the highest and the lowest migration potential, respectively, amongst six different oral cancer cell lines. Subsequent identification of differentially expressed proteins by LC-MS/MS and Western analysis revealed that SERPINB1 (serine protease inhibitor, clade B, member1) was highly expressed in CAL-27, the high-motility oral cancer cells. Semi-quantitative and real-time PCR further confirmed differential expression of SERPINB1 in these two cell lines at mRNA level. To verify the motility-promoting function of SERPINB1 in oral cancer cells, we showed that endogenous expression of SERPINB1 correlated positively with cell migration. Moreover, ectopic expression of SERPINB1 in oral cancer cells, SAS, Ca9-22, CAL-27 and HSC-3, increased cell migration by 25%, 52%, 90% and 100%, respectively. Finally, we found that the expression of SERPINB1 was significantly higher in 5 of 8 (62.5%) oral cancer tissues compared with the matched adjacent normal tissues. Besides, immunohistochemical results indicated over-expression of SERPINB1 in clinicopathologically invasive oral squamous cell carcinoma (OSCC) but not in normal oral mucosa (p<0.01). Together, our findings have provided a possible biomarker for oral cancer metastasis.