Esophageal squamous cell carcinoma (ESCC) is an aggressive type of cancer with high mortality rate in China, largely due to its high invasive and metastatic potential. The purposes of this study are to investigate the potential molecular mechanisms behind the aggressive nature of ESCC and search for new prognostic biomarkers. By employing the quantitative proteomic based strategy, we compared the proteomic profile between three ESCC samples and paired adjacent tissues. After bioinformatics analysis, four candidate proteins were validated in thirteen paired patient samples. Further validation of the key candidate, integrin-linked kinase (ILK), was carried out in one hundred patient samples. The specific inhibitor compound 22 (cpd22) was used to assess the influence of ILK to ESCC cell motility and invasiveness by applying wound-healing and transwell assay. Western blot analysis was performed to elucidate the signaling pathways involved in ILK-mediated ESCC invasion. Total 236 proteins were identified by proteomic analysis. Bioinformatics analysis suggested a key role of the collagen/integrin/ILK signaling pathway during ESCC progression. Further validation indicated that ILK is overexpressed in ESCC tissues and is correlated with poor patient prognosis. Inhibition of ILK kinase activity suppresses proliferation and blocks invasion and migration of ESCC cells. Signaling pathway analysis revealed that ILK regulates AKT phosphorylation on Ser473 but not GSK-3β on Ser9 to promote proliferation and motility of ESCC cells. In conclusion, our results indicated that ILK may play a crucial role in ESCC invasion and metastasis and may serve as a prognostic biomarker and therapeutic target for ESCC.