C‐Jun N‐terminal kinase (JNK) activation is pivotal in the development of nonalcoholic steatohepatitis (NASH). Mixed lineage kinase 3 (MLK) 3 is one of the mitogen activated protein kinase kinase kinase (MAP3K) that mediates JNK activation in the liver. Despite this concept, the role of MLK3 in modulating liver injury during nutrient excess has not been explored. Our aim was to determine if MLK3 deficient mice were protected against high fat high carbohydrate (HFHC) diet‐induced NASH.

We employed eight‐week‐old Mlk3^−/− male C57BL/6J mice, and wild type (WT) mice C57BL/6J as controls. Mice were fed a HFHC or a chow diet adlib for 16 weeks.

Hepatic JNK activating phosphorylation was readily absent in the Mlk3^−/− mice fed the HFHC diet, but not in WT mice. This inhibition of JNK activation was hepatoprotective. Despite a comparable increase in weight gain, hepatic steatosis by histological examination and hepatic triglyceride quantification was reduced in HFHC diet‐fed Mlk3^−/− mice compared with WT mice. In addition, compared with the WT mice, HFHC diet‐fed Mlk3^−/− mice had significantly attenuated liver injury as manifested by reduced ALT levels, hepatocyte apoptosis, markers of hepatic inflammation and indices of hepatic fibrogenesis.

Our results suggest that loss of MLK3 in mice is protective against HFHC diet‐induced NASH, in a weight‐independent fashion, through attenuation of JNK activation. MLK3 is a potential therapeutic target for the treatment of human NASH.