Brown adipose tissue (BAT) plays a critical role in maintaining animal body temperature and energy homeostasis. Growing evidence has demonstrated that guanidinoacetic acid (GAA) is a direct precursor of creatine, which may act as a BAT activator to increase whole-body energy expenditure and metabolism. In this context, the objective of the study was to investigate the effect of dietary GAA supplementation on lipid and glucose metabolism, systemic metabolic homeostasis, and thermogenic function of BAT in middle-aged mice with high-fat diet (HFD)-induced obesity, and to attempt to explore its underlying mechanisms. Our results showed that GAA supplementation could effectively alleviate HFD-induced obesity. Glucose tolerance test (GTT), and insulin tolerance test (ITT) indicated that GAA supplementation significantly restored glucose and insulin tolerance, and reversed the serum levels of TG, TC, LDL-C, and HDL-C in HFD-fed mice. Moreover, GAA treatment contributed to energy expenditure, as reflected by the increased oxygen consumption, carbon dioxide production, respiratory exchange ratio, and heat production. In addition, GAA supplementation increased the expression levels of PPARγ and C/EBPα in the BAT. Meanwhile, GAA supplementation significantly facilitated the thermogenic capacity of BAT by upregulating the expression levels of BAT markers (UCP1, PGC-1α, PRDM16, and Cidea), mitochondrial-related gene (Cox7a), and creatine cycle-related genes, including Gamt, Ckb, Alpl, and Slc6a8. Intriguingly, we observed that dietary GAA supplementation could activate the AMPK/Sirt1 signaling pathway and induce DNA demethylation in BAT. Overall, these results suggest that GAA might have potential as a functional food source for enhancing BAT thermogenesis to combat obesity and related disorders.