Both 14-3-3 proteins (14-3-3s) and Rho proteins regulate cytoskeleton remodeling and cell migration, which suggests a possible interaction between the signaling pathways regulated by these two groups of proteins. Indeed, more and more emerging evidence indicates the mutual regulation of these two signaling pathways. However, all of the data regarding the interaction between Rac1 signaling pathways and 14-3-3 signaling pathways are through either the upstream regulators or downstream substrates. It is not clear if Rac1 could interact with 14-3-3s directly. It is interesting to notice that the Rac1 sequence 68RPLSYP73 is likely a 14-3-3 protein binding motif following the phosphorylation of S71 by Akt. Thus, we hypothesize that Rac1 directly interacts with 14-3-3s. We tested this hypothesis in this research. By using mutagenesis, co-immunoprecipitation (co-IP), Rac1 activity assay, immunoblotting, and indirect immunofluorescence, we demonstrate that 14-3-3s interact with Rac1. This interaction is mediated by Rac1 S71 in both phosphorylation-dependent and -independent manners, but the phosphorylation-dependent interaction is much stronger. Epidermal growth factor (EGF) strongly stimulates the phosphorylation of Rac1 S71 and the interaction between 14-3-3s and Rac1. Mutating S71 to A completely abolishes both phosphorylation-dependent and -independent interactions between 14-3-3s and Rac1. The interaction between 14-3-3s and Rac1 mostly serve to regulate the activity and subcellular localization of Rac1. Among the seven 14-3-3 isoforms, 14-3-3η, -σ, and -θ showed interactions with Rac1 in both Cos-7 and HEK 293 cells. 14-3-3γ also binds to Rac1 in HEK 293 cells, but not in Cos-7 cells. We conclude that 14-3-3s interact with Rac1. This interaction is mediated by Rac1 S71 in both phosphorylation-dependent and -independent manners. The interaction between 14-3-3 and Rac1 mostly serves to regulate the activity and subcellular localization of Rac1. Among the seven 14-3-3 isoforms, 14-3-3η, -γ, -σ, and -θ interact with Rac1.