Recently, increasing evidence reveals that IQGAP1 plays an important role in biological processes of many cancers. Nevertheless, the exact role of IQGAP1 in tumor progression is not unclear, and the function of IQGAP1 in tumor cell biology of ESCC has not been thoroughly investigated. To address these issues, we first investigated the expression of IQGAP1 in ESCC clinical samples by immunohistochemistry, Western blotting and RT-PCR. Different methods showed similar results. EMT is such process which allows epithelial cells to change their phenotype, acquire mesenchymal properties, and increase their capability to migrate. Accumulating evidence has shown that EMT plays an important role in cancer progression and metastasis. PPPA molecular hallmarks of EMT include down-regulation of Ecadherin, which is responsible for the loss of cell-cell adhesion; upregulation of mesenchymal-related proteins N-cadherin, which mediates cell-cell adhesion and modulates cell migration and tumor invasiveness. Interestingly, here we showed that downregulation of IQGAP1 led to a reversion of EMT progress, characterized by decreased expression of mesenchymal markers N-catenin and increased expression of epithelial markers Ecadherin, which was in agreement with IQGAP1 knockdown inhibiting Pladienolide B invasion and metastasis of ESCC cell lines in vitro and in vivo. Indeed, it has been found that IQGAP1 binds directly to Ecadherin and overexpression of IQGAP1 reduces E-cadherinmediated adhesion and facilitates cancer cells to metastasize. These findings present a pivotal role of IQGAP1 in EMT process and tumor promoting function in metastasis of ESCC. IQGAP1, as a scaffold protein, contributes to the transformed cancer cell phenotype by regulating several distinct signaling pathways. Therefore, further research needs to be conducted in order to determine new target protein and molecular mechanism of IQGAP1 contributing ESCC development. In conclusion, our data have provided evidence that IQGAP1 expression is unregulated and correlated with the invasion depth of ESCC. Importantly, by using RNAi technology we have successfully silenced IQGAP1 gene in EC9706 and KYSE150 cells, and for the first time found that downregulation of IQGAP1 expression could inhibit the growth and metastasis of ESCC cells in vitro and in vivo. Further, downregulation of IQGAP1 may lead to a reversion of EMT progress. Therefore, the findings in this study present new insights into the potential role of IQGAP1 in ESCC occurrence and progression, and targeted knockdown of IQGAP1 may represent a promising approach for ESCC treatment. The ability of fluorescence microscopy to probe intracellular processes led to the development of powerful approaches to unveil the diffusive dynamics of biomolecules.