The signal pathway by which 14-3-3ε inhibits cell migration induced by MAPK-activated protein kinase 5 (MK5) was investigated in cultured HeLa cells. Both $in vivo$ and $in vitro$ analyses have revealed that 14-3-3ε interacts with MK5. Other MKs including MK2 and MK3 appeared to fail to interact with 14-3-3ε. 14-3-3ε bound to MK5 inhibits the phosphorylation of HSP27, a known substrate of MK5. Disturbance of actin cytoskeleton organization by 14-3-3ε was shown in transfected cells transiently expressing 14-3-3ε as well as established cells stably expressing 14-3-3ε. Moreover, overexpression of 14-3-3ε resulted in the inhibition of cell migration induced by MK5 overexpression or TNFα treatment. Results in this study suggest that 14-3-3ε bound to MK5 inhibits cell migration by inhibiting the phosphorylation of HSP27 whose phosphorylation regulates F-actin polymerization, actin cytoskeleton organization and subsequent actinfilament dynamics.