Mixed lineage kinase-4 (MLK-4) is an important member of the mixed-lineage family of kinases that regulates the extracellular signal-regulated kinases and c-Jun N-terminal kinase (JNK) signaling pathways. The functions and mechanisms of MLK-4 in cancer initiation and progression have not been well understood. The present study investigated the expression, function and regulatory mechanism of MLK-4 in gastric carcinoma cells. Biochemical data indicated that normal MLK-4 was downregulated, which exerted dominant negative effects on gastric carcinoma cell viability, migration and invasion. The experimental data demonstrated that MLK-4 supplement abrogated activity of these mutants and induced inhibitory effects on gastric carcinoma cell viabilty, migration and invasion in vitro and in vivo. In addition, to determine the regulatory mechanism of MLK-4, its signaling pathway was assessed in gastric carcinoma cancer cells by regulating MLK-4. The present observations indicated that restoring MLK-4 activity by supplemental MLK-4 reduced gastric carcinoma cell colony formation in vitro and suppressed tumor viability, migration and invasion in vivo. The results of the present study indicated that MLK-4 may be a potential protein for targeting gastric carcinoma by suppressing kinases, which may lead to reduction of JNK signaling and enhance therapeutic efficacy in gastric carcinoma.