This study aimed to investigate the location and function of tau isoform of 14-3-3 proteins in human trophoblast. 14-3-3 tau was localized in human cytotrophoblast cells, but not in syncytiotrophoblast cells in both first trimester and term placenta by immunochemistry stain. Forskolin-induced cell fusion (BeWo cells) confirmed that 14-3-3 tau was decreased during trophoblast differentiation. Forskolin-induced differentiation was stimulated by small-interfering (si) RNA induced down-regulation of 14-3-3 tau, contrarily, it was suppressed by plasmid induced upregulation of 14-3-3 tau in BeWo cells. When BeWo cells were treated with 14-3-3 tau siRNA, an increase in protein concentration of cell cycle inhibitor p27kip1 and a decrease in protein concentration of proliferating cell nuclear antigen, as well as activation of the extracellular signal-regulated kinase (ERK) pathway, were also noticed. These findings suggest that 14-3-3 tau might be mediated trophoblast differentiation through cell cycle regulation.