Exosomes are small vesicles derived from the cell membrane and endosomal pathway that are secreted from many different cell types. Exosomes play an important role in mediating communication between cells by delivering proteins, nucleic acids, and lipids. Myristoylation involves the attachment of a myristoyl moiety onto a protein which allows for cell membrane anchoring. Myristoylation mediates Src kinase binding to the plasma membrane and activates cell signaling pathways implicated in cancer. Our study shows that myristoylation promoted the encapsulation of constitutively active Src kinase into exosomes. When Src-encapsulated exosomes were incubated with recipient SYF1 cells, Src kinase was accumulated in a dose- and time-dependent manner. Interestingly, constitutively active mutant Src kinase was not shown to be active within the exosome as well as recipient cells, suggesting a mechanism exists that dephosphorylates Src at its kinase catalytic domain within the exosomes.