Abstract: Focal Adhesion Kinase (FAK) has been indicated in colon cancer development. FAK phosphorylation at Y397 is a critical event in FAK activation. Until recently, FAK phosphorylation in colon cancer has been poorly understood. Therefore, this study conducted western blotting and immunofluorescence analyses to examine the expression and subcellular localization of total FAK and phosphorylated FAK forms in colon cancer cell lines and tumour tissues. This study results showed that differentiated and less aggressive colon cancer cell line; HT-29, expressed lower FAK level compared to non-differentiated and more aggressive colon cancer cell line; HCT 116. Conversely, pY397 was more expressed in HT-29 than in HCT 116, but was more expressed in invasive tissue than in non-invasive and normal tissues. In conclusion, our study suggests a central role of pY397 in the regulation of colon cancer. However, Y397 could be differentially expressed among colon cancer cell lines and tissues.