Bone-related diseases (e.g., osteoporosis) represent a significant health challenge, prompting research for effective therapeutic agents, particularly from natural sources. The edible mushroom, Mycoleptodonoides aitchisonii has attracted interest due to its wide range of biological activities. Cytotoxicity assays revealed no significant toxicity of the M. aitchisonii water extract (MAWE) up to 50 μg/mL. MAWE significantly promoted dose-dependent osteoblast differentiation with ALP activity and mineralization increase by 109.17 % and 23 %, respectively, compared with the differentiation-only group. Moreover, MAWE significantly upregulated osteoblast-related gene expressions, including that of type I collagen (COL1A), osterix (Osx), and osteopontin (OPN). Furthermore, MAWE treatment significantly increased AMPK phosphorylation. This effect was further confirmed by demonstrating that the AMPK inhibitor compound C suppressed AMPK phosphorylation, and subsequent MAWE treatment restored it. In summary, these results demonstrate that MAWE possesses potent osteoblast differentiation-promoting efficacy, primarily through AMPK signaling pathway activation.