Activation of bitter taste receptor member 4 (TAS2R4) signaling alleviates podocyte injury caused by chronic high glucose; however, whether TAS2R4 activation in podocytes can improve diabetic nephropathy (DN) is to be verified. This study aims to confirm the beneficial effects of quinine, a dual human and rodent TAS2R4 agonist, and matrine with a potent anti-inflammatory activity and binding with TAS2R4 via online prediction and receptor docking on DN in vivo and in vitro. In this study, we found that quinine and matrine markedly ameliorated renal dysfunction, as evidenced by decreases in creatinine and urea nitrogen levels in plasma as well as protein excretion in urine, increased podocyte slit diaphragm and adaptor proteins including Nephrin, Podocin, and Zonula occluden 1, and suppressed activations of NF-κB and the NLRP3 inflammasome in the kidney of DN mice. Meanwhile, quinine and matrine activated TAS2R4 signaling, as revealed by increased protein expressions of TAS2R4 and its key downstream molecule phospholipase C β2. Furthermore, quinine and matrine attenuated podocyte injury, activated TAS2R4 signaling, and suppressed the above inflammatory pathways in the high glucose-cultured MPC cells, a mouse podocyte cell line, while the effects of both quinine and matrine were eliminated when TAS2R4 signaling was inhibited by using either a TAS2R4 blocker abscisic acid or a Gβγ inhibitor Gallein. In summary, quinine and matrine alleviated DN in mice through activation of TAS2R4 signaling in podocytes, which was achieved by inhibiting the activation of NF-κB mediated NLRP3 inflammasome in the kidney. Moreover, TAS2R4 could be a drug target.