Thymosin beta 4 (T beta 4) is a 43-amino-acid peptide involved in many biological processes. However, the precise molecular signaling mechanism(s) of T beta 4 in cell invasion and migration remain unclear. In this study, we show that T beta 4 was significantly overexpressed in colorectal cancer tissues compared to adjacent normal tissues and high levels of T beta 4 were correlated with stage of colorectal cancer, and that T beta 4 expression was associated with morphogenesis and EMT. T beta 4-upregulated cancer cells showed increased adhesion, invasion and migration activity, whereas T beta 4-downregulated cells showed decreased activities. We also demonstrated that T beta 4 interacts with ILK which promoted the phosphorylation and activation of AKT, the phosphorylation and inactivation of GSK3 beta, the expression and nuclear localization of beta-catenin, and integrin receptor activation. These results suggest that T beta 4 is an important regulator of the ILK/AKT/beta-catenin/Integrin signaling cascade to induce cell invasion and migration in colorectal cancer cells, and is a potential target for cancer treatment. (C) 2014 Elsevier Inc. All rights reserved.