CSK-Homologous Kinase (CHK) is a potential tumour suppressor in many types of cancer. It exerts its tumour suppressor action mainly by inhibiting the oncogenic Src-family tyrosine kinases (SFKs). CHK inhibits SFKs by phosphorylating the conserved C-terminal regulatory tyrosine near the C-terminus of SFKs and by employing a non-catalytic inhibitory mechanism to inhibit SFKs. This non-catalytic mechanism involves direct binding of CHK to the kinase domain of SFK to form a stable complex, which is sufficient to inhibit SFK activity. As CSK (a homolog of CHK) lacks this non-catalytic mechanism, this property is unique to CHK. Hence, this project aims to decipher the structural basis of the non-catalytic inhibitory mechanism of CHK. The crystal structure complex of the kinases of both CSK and SFK member (c-Src) shows five basic residues of CSK form ionic and hydrophobic interactions with the negatively charged and hydrophobic residues near the C-terminal tail of c-Src. Since these five basic residues are conserved in CSK and CHK, it is likely that they also participate in binding of CHK to the SFK. Results of our studies indicate that all five residues including Arg-276, Arg-278 and Arg-280 in the αD helix  and Arg-382 and Lys-387 in the αF-αG loop of the CHK are determinants governing allosteric inhibition of SFKs by CHK. Arg-382 and Lys-387 play significant role in the tight binding of CHK to SFKs. Since CHK contains a C-terminal tail that is eighteen residues longer than that of CSK, we investigated the effect of truncation of the C-terminal tail on the ability of CHK to bind and inhibit SFKs.Our data shows that the C-terminal tail of CHK contains determinant(s) governing allosteric inhibition of SFKs. Since Arg-278 was recently discovered to be mutated in colon cancer, our results suggest that this mutation likely contributes to aberrant activation of the oncogenic SFKs and in turn colon cancer formation and progression by abolishing the non-catalytic inhibitory mechanism of CHK.