14-3-3 proteins are ubiquitously expressed dimeric adaptor proteins that have emerged as key mediators of many cell signaling pathways in multiple cell types. Its effects are mainly mediated by binding to selective phosphoserine/threonine proteins. The importance of 14-3-3 proteins in cancer have only started to become apparent and its exact role in cancer progression as well as the mechanisms by which 14-3-3 proteins mediate cancer cell function remain unknown. While protein 14-3-3σ is widely accepted as a tumor suppressor, 14-3-3ζ, β and γ isoforms have been shown to have tumor promoting effects. Despite the importance of 14-3-3 family in mediating various cell processes, the exact role and mechanism of 14-3-3ζ remain unexplored. In the current study, we investigated the role of protein 14-3-3ζ in prostate cancer cell motility and transendothelial migration using biochemical, molecular biology and electric cell-substrate impedance sensing approaches as well as cell based functional assays. Our study indicated that expression with wild-type protein 14-3-3ζ significantly enhanced Rac activity in PC3 cells. In contrast, expression of dimer-resistant mutant of protein 14-3-3ζ (DM-14-3-3) inhibited Rac activity and associated phosphorylation of p21 activated kinase-1 and 2. Expression with wild-type 14-3-3ζ or constitutively active Rac1 enhanced extracellular matrix recognition, lamellipodia formation, cell migration and trans-endothelial migration by PC3 cells. In contrast, expression with DM 14-3-3ζ or DN-Rac1 in PC3 cells significantly inhibited these cell functions. Our results demonstrate for the first time that 14-3-3ζ enhances prostate cancer cell-matrix interactions, motility and transendothelial migration in vitro via activation of Rac1-GTPase and is an important target for therapeutic interventions for prostate cancer.