The family of 14-3-3 proteins is implicated with many biological activities by binding to other proteins. It consists of seven highly conserved isoforms (β, γ, ε, ζ, η, σ, and τ) and most of them are identified as oncogenes in various types of cancer except 14-3-3σ, a well-known tumor suppressor. In non-small cell lung cancer, we found that overexpression of 14-3-3 proteins increased cell growth besides 14-3-3σ, which inhibited cancer cell viability. To clarify the suppressor characteristic of 14-3-3σ divergent from others, the protein alignment of 14-3-3 family found that the major difference between the other oncogenic members and 14-3-3σ was a substitution at amino acid 180 from Tyr to His by which a SH2-binding motif (YYEI) is mutated. Thus, we generated an H180Y 14-3-3σ mutant by site-direct mutagenesis and investigated the impact of this amino acid change on tumor suppressive activity. First, we found that overexpression of 14-3-3σ decreased the abilities of cancer cell invasion. Surprisingly, the H180Y mutant not only enhanced cell invasiveness but also promoted cell viability. Meanwhile, the interaction between H180Y mutant and Src was stronger than that of wild type. The fact indicates one amino acid substitution switches 14-3-3σ from tumor suppressor to oncogene. Hence, the YYEI motif might be important for the oncogenic activity of other 14-3-3 member proteins. Indeed, we demonstrated that 14-3-3ζ interacted with Src through Tyr178, which is crucial for the binding of 14-3-3ζ with Src-SH2 domain. Owing to the importance of Tyr178 in the 14-3-3ζ/Src interaction, we introduced the Y178F 14-3-3ζ mutant to lung cancer cells and confirmed that Tyr178 is important for the increase of invasion and viability. Nevertheless, we found no single nucleotide polymorphism (SNP) of 14-3-3σ in clinical samples of NSCLC patients. We also failed to design effective synthetic peptides based on YYEI and HYEI to kill cancer cells. Fortunately, we revealed an amino acid switch of 14-3-3σ from YYEI to HYEI during evolutionary progression, showing the special features of YYEI motif in 14-3-3 family. Taken together, our findings suggested that YYEI motif is essential for 14-3-3 proteins to interact with Src and to regulate Src-mediated cell functions.