Annexin A1 (ANXA1) acts either as a tumor suppressor or an oncogene in different tumor types. Several clinical studies revealed that the expression of ANXA1 is associated with the pathologic differentiation grade in oral squamous cell carcinoma (OSCC) patients. However, the direct function of ANXA1 in OSCC progression has remained to be fully clarified. The present study was designed to investigate the role of ANXA1 in OSCC cell proliferation and invasion in vitro. Furthermore, whether ANXA1 was involved in transforming growth factor β1 (TGFβ1)/epidermal growth factor (EGF)-induced epithelial-mesenchymal transition (EMT) in OSCC was explored. Tca-8113 and SCC-9 cells were transfected with ANXA1-pcDNA3.1 plasmid to overexpress ANXA1. Subsequently, cell proliferation and invasion were examined using MTT and Transwell-Matrigel invasion assays. TGFβ1 and EGF were used to induce EMT in Tca-8113 and SCC-9 cells, and the expression of epithelial (E)-cadherin, neural (N)-cadherin and vimentin was determined by western blot analysis. The results demonstrated that ANXA1 overexpression induced a significant decrease of cell growth and invasiveness in Tca-8113 and SCC-9 cells. The expression of E-cadherin was significantly increased, while the expression of vimentin and N-cadherin was significantly decreased in ANXA1-overexpressing Tca-8113 and SCC-9 cells. ANXA1 expression was significantly decreased in TGFβ1/EGF-treated cells. Furthermore TGFβ1/EGF-induced EMT in OSCC cell lines was attenuated by ANXA1 overexpression. In conclusion, to the best of our knowledge, the present study was the first to evidence that ANXA1 inhibits OSCC cell proliferation and invasion in vitro. TGFβ1/EGF-induced EMT was reversed by ANXA1 in OSCC. ANXA1 was suggested to be a potential marker for OSCC as well as a novel treatment.