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Currently, Bifidobacterium, Lactobacillus, and Streptococcus thermophilus (BLS) are widely recognized as the crucially beneficial bacteria in the gut. Many preclinical and clinical studies have shown their protective effects against non-alcoholic fatty liver disease (NAFLD). However, whether gestational BLS supplementation could alleviate NAFLD in the offspring is still unknown. Kunming mice were given a high-fat diet (HFD) for 4 weeks before mating. They received BLS supplementation by gavage during pregnancy. After weaning, offspring mice were fed with a regular diet up to 5 weeks old. Gestational BLS supplementation significantly increased the abundance of Actinobacteriota, Bifidobacterium, and Faecalibaculum in the gut of dams exposed to HFD. In offspring mice exposed to maternal HFD, maternal BLS intake significantly decreased the ratio of Firmicutes to Bacteroidetes as well as the relative abundance of Prevotella and Streptococcus, but increased the relative abundance of Parabacteroides. In offspring mice, maternal BLS supplementation significantly decreased the hepatic triglyceride content and mitigated hepatic steatosis. Furthermore, maternal BLS supplementation increased the glutathione content and reduced malondialdehyde content in the liver. In addition, mRNA and protein expression levels of key rate-limiting enzymes in mitochondrial β-oxidation (CPT1α, PPARα, and PGC1α) in the livers of offspring mice were significantly increased after gestational BLS supplementation. Thus, gestational BLS supplementation may ameliorate maternal HFD-induced steatosis and oxidative stress in the livers of offspring mice by modulating fatty acid β-oxidation., (© 2024 Institute of Food Technologists.)