Thymosin β(4) (Tβ(4)) overexpression increases cell migration and tumor metastasis. Hence, understanding the mechanism of cancer cell migration induced by Tβ(4) may provide means to inhibit their metastasis. We demonstrated higher Rac1 activities and expression levels of IQGAP1 and ILK in highly migrated Tβ(4)-overexpressing SW480 cells. In addition, IQGAP1 formed a complex with ILK and knockdown of Tβ(4) simultaneously reduced ILK and IQGAP1 protein levels as well as their migration ability. These findings suggest that Tβ(4) increases migration of colon cancer cells via activating Rac1 by elevating IQGAP1/ILK complexes and IHC results illustrated a similar mechanism occurring in vivo.