Anemarrhena asphodeloides Bunge is widely used in China for the treatment of diabetes and the polyphenol components are responsible for its anti-diabetic action. This study aimed to investigate the effect of total polyphenol of Anemarrhena asphodeloides (TPAA) on endothelial dysfunction and to elucidate underlying mechanisms. We stimulated endothelial cells with advanced glycation end products (AGEs) to establish the model of endothelial dysfunction in vitro and observed the effect of TPAA (10, 30, or 100 μg/mL) on AMP-Kinase (AMPK) activation implicated in regulation of nitric oxide (NO) and endothelin-1 (ET-1) production. Meanwhile, nuclear factor-κB (NF-κB) activation, intracellular reactive oxygen species (ROS) production, mitochondrial membrane potential (ΔΨm) and eNOS expression were investigated by western blot, fluorescence microscopy and real time-quantitative PCR analysis, respectively. Total polyphenol of Anemarrhena asphodeloides enhanced AMPK phosphorylation and promoted the basal NO production along with the inhibition of ET-1 secretion in endothelial cells. TPAA inhibited NF-κB activation by attenuating p65 phosphorylation and suppressed ROS production, well demonstrating its action in inhibition of ROS-associated inflammation in the endothelium. Meanwhile, TPAA protected mitochondrial function and endothelial homeostasis against AGEs insult by restoring ΔΨm and mRNA expression of eNOS. AGEs stimulation inhibited AMPK activation and induced the loss of NO production together with increased secretion of ET-1, but these changes were reversed by TPAA in a concentration-dependent manner. Compound C, an AMPK inhibitor, attenuated the effects of TPAA mentioned above, indicating the involvement of AMPK. Total polyphenol of Anemarrhena asphodeloides inhibited AGEs-induced ROS-associated inflammation and ameliorated endothelial dysfunction through beneficial regulation of AMPK activation.