14-3-3 family consists of seven highly conserved isoforms and most of them are identified as oncogenes in various types of cancer except for 14-3-3σ, a well-known tumor suppressor. To clarify the suppressor characteristic of 14-3-3σ divergent from others, we found that the major difference between the other oncogenic members and 14-3-3σ by protein alignment was an amino acid substitution (Y180H) by which the SH2-binding motif (YYEI) is disrupted and cannot be phosphorylation. Thus, we generated a H180Y 14-3-3σ mutant and investigated the impact of the acquired YYEI motif on the tumor suppression. First, we found that 14-3-3σ decreased cancer invasion. Surprisingly, the H180Y mutant not only enhanced cell invasion but increased cell viability. Meanwhile, the interaction between H180Y mutant and Src was higher than wild type. It indicates one amino acid substitution switches 14-3-3σ from tumor suppressor to oncogene. Hence, the YYEI motif might be important for the oncogenicity of other 14-3-3 proteins. Indeed, we demonstrated that 14-3-3ζ interacted with Src through Y178 phosphorylation, which is crucial for the binding of 14-3-3ζ with Src-SH2 domain. Owing to the importance of Y178 phosphorylation in the 14-3-3ζ/Src interaction, we introduced the Y178F 14-3-3ζ mutant to lung cancer cells and confirmed that Y178 phosphorylation is important for the increase of invasion and viability. Furthermore, we also revealed an amino acid switch of 14-3-3σ from YYEI to HYEI during evolutionary progression. Taken together, our findings suggest that YYEI motif is essential for 14-3-3 proteins to interact with Src and to regulate Src-mediated cell functions.