Pueraria lobata (Willd.) Ohwi, known as kudzu and used as a "longevity powder" in China, is an edible plant which is rich in flavonoids and believed to be useful for regulating blood sugar and treating diabetes, although the modes of action are unknown. Here, a total of 53 flavonoids including 6 novel compounds were isolated from kudzu using multidimensional preparative liquid chromatography. The flavonoid components were found to lower blood sugar levels, promote urine sugar levels in mice, and reduce the urine volume. Molecular docking and in vitro assays suggested that the antidiabetic effect of kudzu was attributed to at least three targets: sodium-dependent glucose transporter 2 (SGLT2), protein tyrosine phosphatase-1B (PTP1B), and alphaglucosidase (AG). This study suggests a possible mechanism for the antidiabetic effect that may involve the synergistic action of multiple active compounds from kudzu.