Objective To investigate the molecular mechanism of palmitoylation modification in regulating the activity of non-receptor tyrosine kinase Fyn. Methods The intracellular Fyn activity was detected by applying fluorescence resonance energy transfer (FRET) technology, and the mechanism was investigated by combining with Fyn palmitoylation deficiency and C-terminal Src kinase ( CSK ) plasmid co-expression. ResultsExperimental data showed that single loss of either of ( C3, C6) palmitoylation sites resulted in higher Fyn activity, and C6 seemed more significant. It is known that CSK membrane translocation occurred after activation. FRET assay confirmed that CSK could down-regulate the activity of Fyn in cells, but could not effectively regulate the activity of Fyn(GSS) with the loss of palmitoylation sites. Conclusions The results in this study support the hypothesis on Fyn regulation by spatial localization, namely, non-palmitoylated Fyn (GSS) is less effective in the inhibitory regulation by CSK on cell membrane, thus promoting constitutive high activity expression