The tumor suppressor gene FHIT is inactivated by genetic and epigenetic changes in the majority of common human cancers. The human Fhit protein undergoes phosphorylation on tyrosine residue 114 by Src and related kinases both in vitro and in vivo. Src is a key cytoplasmic tyrosine kinase downstream to several growth factor receptors, including those of the EGF receptor family, which are overexpressed and activated in about one-third of human breast and ovarian carcinomas. However, the biological significance of Fhit phosphorylation by Src has remained elusive. In the present study, we demonstrate that FHIT acts as a checkpoint in cell proliferation mediated by activated tyrosine kinase receptors that recruit Src. Activation of EGF receptor family members induced Fhit phosphorylation by Src and the subsequent proteasome degradation of the phosphorylated Fhit protein. Indeed, the use of the Fhit mutant Y114F, which carries a phenylalanine instead of a tyrosine at position 114, unable to be phosphorylated on tyrosine 114 by Src, prevents Fhit degradation. Moreover, Fhit protein reduction is transient and occurs in a specific temporal window. During the signaling pathway of activated tyrosine kinase receptors, the phosphorylation of Fhit induces its degradation and the subsequent reduction in Fhit protein levels allows the transmission of the mitogenic signal; immediately thereafter, Fhit protein levels are restored. Such a scenario would suggest a key role for Fhit in the balance of proliferation/survival/apoptosis signals.