Src activation has been associated with colon cancers but the mechanism underlying Src activation is largely unknown. Csk-homologous kinase (CHK) can inhibit the kinase activity of certain Src kinase family members in vitro by phosphorylating the C-terminal tyrosine and by a non-catalytic mechanism. CHK was previously reported to be expressed primarily in brain and hematopoietic cells. We report herein that CHK is also expressed in normal colon cell lines. Furthermore, CHK protein levels are significantly decreased in various colon cancer cell lines and the decrease correlates with the increased specific activity of Src in these cell lines, while the level of the other Src inhibitory kinase, C-terminal Src kinase, is not significantly changed. CHK is also expressed in normal colon tissues but its expression level is decreased in colon cancer tissues collected from the same patients. Immunofluorescence microscopy shows that CHK colocalizes with Src in normal colon FHC cells. Overexpression of CHK in colon cancer cells results in inactivation of Src without phosphorylating Y530 at its C-terminus. In addition, CHK suppresses anchorage-independent cell growth and cell invasion of colon cancer cells. These results reveal a potentially important role for CHK in Src activation and tumorigenicity in colon cancer cells.