Smilax glabra Roxb. flavonoids (SGF) are the rhizome extracts of Smilax glabra Roxb. and possesses pharmacological activity, prior studies have demonstrated SGF’s cardioprotective potential, specifically its capacity to inhibit pathological cardiac hypertrophy. Building upon this evidence, this study investigated whether SGF exerts protective effects against D-galactose-induced cardiac alterations and elucidated its underlying molecular mechanisms. D-galactose was employed to induce aging-like changes in C57BL/6J mice and H9c2 cell. The cardioprotective effects of SGF against D-galactose-induced myocardial alterations were investigated, with particular emphasis on mitochondrial ultrastructural integrity, expression of key factors in the PGC-1α/NRF1/TFAM axis and PINK1/Parkin signaling pathways, in relation to AMPK signaling. Additionally, in vitro, the AMPK inhibitor Compound C and the AMPK agonist AICAR were used to examine whether the cardioprotective effect of SGF was associated with AMPK. In vivo, SGF administration significantly attenuated D-galactose-induced cardiac hypertrophy, ameliorated myocardial fibrosis and ROS overproduction, preserved mitochondrial ultrastructure, enhanced AMPK phosphorylation (increased p-AMPKα/AMPK ratio), and upregulated the expression of key proteins involved in the PGC-1α/NRF1/TFAM axis and PINK1/Parkin signaling pathways. In vitro, SGF effectively suppressed D-galactose-induced senescence-like changes in H9c2 cells, these beneficial effects were abolished upon co-treatment with Compound C, and the action of SGF was similar to that of AICAR. These results suggested that SGF attenuated D-galactose-induced cardiac hypertrophy and preserves mitochondrial structure. These effects were accompanied by increased AMPK phosphorylation and increased expression of proteins related to PGC-1α/NRF1/TFAM axis and PINK1/Parkin signaling pathway. These changes were consistent with an AMPK-associated pattern and may contribute to the cardioprotective effects of SGF via effects on mitochondrial function.