Val-Val-Tyr-Pro (VVYP) peptide is one of the main active components of Globin digest (GD). Our previous studies indicated that VVYP could protect against acetaminophen and carbon tetrachloride-induced acute liver failure in mice and decrease blood lipid level. However, the effects and underlying mechanisms of VVYP in the treatment of non-alcoholic steatohepatitis (NASH) have not been discovered. Our present study was designed to investigate the preventive effect of VVYP on NASH and its underlying specific mechanisms. We found that VVYP inhibited the cytotoxicity and lipid accumulation in L-02 cells that were exposed to a mixture of free fatty acid (FFA). VVYP effectively alleviated the liver injury induced by methionine-choline-deficient (MCD) diet, demonstrated by reducing the levels of serum alanine aminotransferase (ALT)/aspartate aminotransferase (AST)/triglycerides (TG)/non-esterified fatty acids (NEFA) and improving liver histology. VVYP decreased expression levels of lipid synthesis-related genes and reduced levels of the proinflammation cytokines in the liver of mice fed by MCD diet. Moreover, VVYP inhibited the increased level of LPS and reversed the liver mitochondria dysfunction induced by MCD diet. Meanwhile, VVYP significantly increased the abundance of beneficial bacteria such as Eubacteriaceae, coriobacteriacease, Desulfovibrionaceae, S24-7 and Bacteroidia in high-fat diet (HFD)-fed mice, however, VVYP reduced the abundance of Lactobacillus. Moreover, VVYP conferred the protective effect of intestinal barrier via promoting the expression of the mucins and tight junction (TJ)-associated genes and inhibited subsequent liver inflammatory responses. These results indicated that the protective role of VVYP on NASH is mediated by modulating gut microbiota imbalance and related gut-liver axis activation. VVYP might be a promising drug candidate for NASH. © 2021 The Authors. Journal of Cellular and Molecular Medicine published by Foundation for Cellular and Molecular Medicine and John Wiley & Sons Ltd.