MLK4 is a member of the mixed lineage family of kinases activated by environmental stress, cytokines and growth factors. These kinases lie upstream of the MKK4/7 and MKK3/6 which are critical regulators of the JNK and p38 pathways. Mutations have been identified in MLK4 in various human cancers, especially in colon and lung cancer at a frequency of 6.8% and 2.6%, respectively. Bioinformatics analysis suggests that majority of the mutations in MLK4 are predicted to be pathological (PMUT) or cancer-associated (CanPredict), and several occur at residues critical for kinase activity (for example, in the DFG and HRD motifs). The functional consequences of these mutations have not been elucidated so far. We generated MLK4 cancer mutants by site-directed mutagenesis and compared the activation of these mutants against WT, constitutively active and kinase dead MLK4. We monitored activation of the MKK4/7/JNK pathway and we observed that a majority of mutations are loss-of-function (LOF). To determine if these mutations are critical for maintenance of tumorigenic phenotypes we used the LoVo cell line harbouring an inactivating mutation in MLK-4 (R470C) and an activating mutation in KRas (G13D). The reconstitution of WT and constitutively active MLK-4 in LoVo cells induced selective activation of MKK4/MKK7/JNK pathway without any effects on the Raf/MEK/ERK pathway. These data suggest that LOF mutations may enhance tumorigenesis by decreasing signalling through the MKK4/7 pathway in cells with activating mutations in components of the Raf/MEK/ERK pathway. In summary our data convey a complex picture where a majority of MLK4 mutations alter kinetic activity to modify cancer-signalling pathways dependent upon the presence of other oncogenic mutations.