The effects of Cucumaria frondosa polysaccharides (CFPs) on renal interstitial fibrosis by regulating the phosphatidylinositol-3-hydroxykinase/protein kinase-B/nuclear factor-κB (PI3K/AKT/NF-κB) signaling pathway were investigated in vivo and in vitro in this research. The common unilateral urethral obstruction (UUO) model was used to examine the renoprotective effect and its mechanism in vivo. Compared to the UUO group, CFP administration could ameliorate renal function, inhibit inflammation and fibrosis, and reduce the deposition of the extracellular matrix and epithelial–mesenchymal transition. Mechanistic results indicated that CFPs could inhibit the expression of the total protein of PI3K and the conversion of the AKT and NF-κB p65 phosphorylated proteins, thereby inhibiting the transduction of the PI3K/AKT/NF-κB pathway. In addition, CFP treatment could improve inflammation and fibrosis in HK-2 cells induced by TGF-β1, and its in vitro mechanism was also verified to inhibit the PI3K/Akt/NF-κB signaling pathway. Overall, these results showed that CFP could alleviate renal interstitial fibrosis related to the PI3K/AKT/NF-κB signaling pathway.