Xanthine oxidase (XO) represents a key therapeutic target for the treatment of hyperuricemia. Although dietary anthocyanins are known to ameliorate hyperuricemia, their structure–activity relationship (SAR) concerning XO inhibition has not been fully elucidated. This study systematically evaluated the inhibitory effects of anthocyanin extracts derived from various dietary sources—including fruits, vegetables, cereals, and edible flowers—on XO activity, uric acid (UA) levels, and hyperuricemia-associated biomarkers. Using inhibition kinetics, spectroscopic techniques, and molecular simulations, we confirmed the direct binding of anthocyanins to XO and identified delphinidin-3-O-sambubioside (D3S) as the most potent inhibitor. In vivo experiments showed that anthocyanin extract from kale significantly suppressed XO activity and improved renal function in hyperuricemic mice. Transcriptomic and qPCR analyses further revealed that anthocyanins modulate the PI3K-Akt signaling pathway, specifically through downregulation of PIK3R5. These findings highlight the potential of specific anthocyanin structures, particularly D3S, as effective natural XO inhibitors for the management of hyperuricemia.