Abstract Actin depolymerizing factor (ADF)/cofilin family proteins are key regulators of actin filament turnover and cytoskeleton reorganization. The role of cofilin‐1 in cell motility has been demonstrated in several cell types but remained poorly documented in the case of colon cancer. In addition, the putative function of destrin (also known as ADF) had not been explored in this context despite the fact that it is expressed in all colon cancer cell lines examined. We were therefore prompted to evaluate the respective contributions of these proteins to the invasive properties of the human colon cancer Isreco1 cell line, which expresses a comparatively high destrin/cofilin ratio. Reduction of cofilin‐1 or destrin expression in Isreco1 cells using RNA interference led to an increase of the number of multinucleated cells and altered polarized lamellipodium protrusion and distribution of paxillin‐containing adhesions. Both cofilin‐1 and destrin silencing enhanced cell adhesion to extracellular matrix components. However, only destrin appeared to be required for cell migration on collagen I and for cell invasion through Matrigel in response to the proinvasive neuroendocrine peptide bombesin. This differential functional involvement was supported by a destrin‐dependent, cofilin‐independent phosphorylation of p130Crk‐associated substrate (p130Cas) upon cell adhesion to collagen I or Matrigel. Taken together, our results suggest that destrin is a significant regulator of various processes important for invasive phenotype of human colon cancer Isreco1 cells whereas cofilin‐1 may be involved in only a subset of them. © 2007 Wiley‐Liss, Inc.