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Nonalcoholic fatty liver disease (NAFLD) is a common chronic liver disease with many influencing factors. With the increasing role of the gut–liver axis in various liver diseases, research on the prevention and treatment of NAFLD with probiotics is increasing. In the present study, a <i>Bifidobacterium animalis</i> subsp. strain, <i>B. lactis</i> SF, was isolated from the feces of healthy infants and characterized by sequencing of the 16S rDNA. A systematic probiotic evaluation was carried out, and a diet-induced mouse model was constructed to study the effect and mechanism of <i>B. lactis</i> SF on diet-induced NAFLD. Results show that <i>B. lactis</i> SF has excellent gastrointestinal fluid tolerance and intestinal colonization, and strong antibacterial and antioxidant capabilities. In vivo, <i>B. lactis</i> SF modulated intestinal flora, restored the intestinal barrier, and inhibited LPS entrance into the portal circulation, which subsequently inhibited the TLR4/NF-κB and modulated the PI3K-Akt/AMPK signaling pathway, attenuated the inflammatory response, and reduced lipid accumulation. In addition, <i>B. lactis</i> SF attenuated oxidative stress and further alleviated autophagy, resulting in an ameliorative effect on NAFLD. Therefore, our study provides a new dietary method for the treatment of NAFLD.