Non-alcoholic fatty liver disease (NAFLD) is the leading cause of chronic liver diseases worldwide, necessitating urgent novel oral treatments. In this study, β-cell expansion factor A (BefA) was evaluated in a murine NAFLD model induced by high-fat diet (HFD). Our results revealed that BefA significantly reduced body weight (36.58 ± 1.55 g vs. 42.30 ± 1.96 g, p < 0.01), fat mass-to-body weight ratio (0.023 ± 0.019 vs. 0.300 ± 0.019, p < 0.05), liver weight (1.90 ± 0.07 g vs. 2.31 ± 0.21 g, p < 0.05), and liver function parameters (ALT, AST, ALP levels reduced, p < 0.05). Notably, BefA reversed the pathological features of NAFLD, decreasing hepatic steatosis score from 3.67 ± 0.47 to 1.67 ± 0.47 (p < 0.01). Mechanistically, BefA activated the AMPK signaling pathway, resulting in the suppression of lipogenic gene transcription (ACC, FASN, SREBP-1c) and the enhancement of fatty acid oxidation (CPT-1, PPAR-α). However, AMPK inhibitor and broad-spectrum antibiotics significantly attenuated the benefits observed with BefA treatment, increasing body weight, fat-to-body weight ratio, and liver weight (p < 0.05). Similar detrimental effects were also observed in liver function indices and histopathological characteristics. These findings underscore the pivotal role of both gut microbiota modulation and AMPK signaling in BefA's therapeutic efficacy, making it a promising multitargeted approach for NAFLD treatment.