Pituitary adenoma (PA) is mostly benign intracranial tumor, but it also displays invasive growth characteristics and provokes challenging clinical conditions. S100A9 protein enhances tumor progression. In this study, we firstly demonstrated that both intracellular and extracellular S100A9 promoted the expression of Vimentin and Intercellular cell adhesion molecule-1 (ICAM-1), coupled with reduced E-cadherin in PA. As a result, PA acquired the phenotype of Epithelial-Mesenchymal Transition (EMT), leading to proliferation, cell cycle progression, migration and invasion. In addition, we indicated S100A9-induced EMT was mediated by activation of AKT1. Furthermore, immunohistochemistry showed that S100A9 expression was higher in invasive PA than that in non-invasive PA. These data extended our understanding for the effects of S100A9 on PA invasion and contributed to further development of a promising therapeutic target for invasive PA.