Obesity is a chronic metabolic disease worldwide and is considered a major health problem in contemporary society. Lactobacillus acidophilus have demonstrated beneficial effects on obesity, but the specific mechanism of how it exerts beneficial effects has not been elucidated. Here, we found that L. acidophilus JYLA-126 had good biological properties for intestinal health, such as antioxidation, acid tolerance, bile salt tolerance, antimicrobial activity, and gut colonization. We further identified that supplementation of L. acidophilus JYLA-126 obese mice possessed a dose-dependent amelioration of body weight, intestinal imbalance, and metabolic disorders compared to HFD-induced mice. Mechanistically, the excellent slimming effect of L. acidophilus JYLA-126 was achieved mainly by reversing HFD-induced gut dysbiosis, inhibiting inflammatory factors and balancing the homeostasis of the gut-liver axis. Specifically, L. acidophilus JYLA-126 improved hepatic glycogen synthesis, lowered oxidative stress, and facilitated lipid metabolism by regulating AMPK signaling pathway-related protein expression to restore the overall metabolic level. Accordingly, L. acidophilus JYLA-126 promoted energy uptake efficiency in obese mice, resulting in significant expression of uncoupling protein 1 (UCP1) protein in brown adipose tissue (BAT), and markedly reduced the size of adipocytes. These findings indicate that the anti-obesity activity of L. acidophilus JYLA-126 correlates with activation of the AMPK signaling pathway through improved gut-liver interactions.