Mesangial proliferative glomerulonephritis (MPGN) is the most common type of chronic kidney disease in China, characterized by mesangial cell proliferation and inflammatory response. Paeoniflorin, an effective composition extracted from Radix Paeoniae Alba, has been used for various kinds of kidney diseases. However, there are no studies reporting the effects of paeoniflorin on MPGN. The present study aims to investigate whether paeoniflorin plays a role in MPGN and confirm the underlying molecular mechanisms. Our results manifested that paeoniflorin strongly restrained 24 h urinary protein and promoted renal function and dyslipidemia in a MPGN rat model. Moreover, paeoniflorin attenuated mesangial cell proliferation and inflammation both in MPGN rats and human mesangial cells (HMCs) treated with lipopolysaccharide (LPS). In detail, paeoniflorin decreased the number of mesangial cells and expressions of proliferation marker Ki67 in MPGN rats. Paeoniflorin also inhibited HMC proliferation and blocked cell cycle progression. In addition, the contents of inflammatory factors and the expressions of macrophage marker iNOS were decreased after paeoniflorin treatment. Furthermore, we found that the protective effect of paeoniflorin was accompanied by a strong inhibition of the phosphatidylinositol 3-kinase (PI3K)/AKT/glycogen synthase kinase (GSK)-3 beta pathway. Paeoniflorin enhanced the inhibitory effect of PI3K inhibitor LY294002 and suppressed the activated effect of PI3K agonist insulin-like growth factor 1 (IGF-1) on PI3K/AKT/GSK-3 beta pathway. In conclusion, these results demonstrated that paeoniflorin ameliorates MPGN by inhibiting mesangial cell proliferation and inflammatory response through the PI3K/AKT/GSK-3 beta pathway.