Mixed lineage kinase (MLK) 4, or MLK4, is a member of the MLK family of mitogen-activated protein kinase kinase kinases (MAP3Ks). Typically, MAP3Ks function to activate the mitogen-activated protein kinase (MAPK)-signaling pathways and regulate different cellular responses. However, here we report that MLK4β, unlike the other MLKs, negatively regulates the activities of the MAPKs, p38, c-Jun N-terminal kinase and extracellular signal-regulated kinase, and the MAP2Ks, MEK3 and 6. Our results show that MLK4β inhibits sorbitol- and tumor necrosis factor-induced activation of p38. Furthermore, MLK4β interacts with another MLK family member, MLK3, in HCT116 cells. Exogenous expression of MLK4β inhibits activation of MLK3 and also blocks matrix metalloproteinase-9 gelatinase activity and invasion in SKOV3 ovarian cancer cells. Collectively, our data establish MLK4β as a novel suppressor of MLK3 activation, MAPK signaling and cell invasion.