Objective: Fyn is a non-receptor tyrosine kinase, which has an important role in regulating T cell immune response, neuronal development, and cell adhesions. Previous study has reported that N-terminal (C3, C6) palmitoylation regulates Fyn activity, but the molecular mechanism is lacking. Methods: In this study, by applying fluorescence resonance energy transfer (FRET) technology to detect intracellular Fyn activity, the mechanism was investigated combining with Fyn palmitoylation deficiency and CSK (C-terminal Src kinase) plasmid co-expression. Results: Experimental 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. Conclusion : Combined with previous studies, these results support the hypothesis on Fyn regulation by spatial localization that non-palmitoylated Fyn(GSS) is less effective in the inhibitory regulation by CSK on the cell membrane, thus promoting constitutive high activity expression.