Patients with chronic kidney disease (CKD) lack efficacious treatment. Jian-Pi-Yi-Shen formula (JPYSF) has demonstrated significant clinical efficacy in treating CKD for decades. However, its renoprotective mechanism has not been fully elucidated. This study aimed to determine whether JPYSF could delay renal fibrosis progression in CKD by restoring nicotinamide adenine dinucleotide (NAD+) biosynthesis.Adenine-diet feeding was used to model CKD in C57BL/6 mice. JPYSF was orally administered for 4 weeks. Human proximal tubular epithelial cells (HK-2) cells were stimulated with transforming growth factor-β1 (TGF-β1) with or without JPYSF treatment. Renal function of mice was assessed by serum creatinine and blood urea nitrogen levels. Renal histopathological changes were assessed using Periodic acid-Schiff and Masson's trichrome staining. Cell viability was assessed using a cell counting kit-8 assay. NAD+ concentrations were detected by a NAD+/NADH assay kit. Western blotting, immunohistochemistry, and immunofluorescence were employed to examine fibrosis-related proteins and key NAD+ biosynthesis enzymes expression in the CKD kidney and TGF-β1-induced HK-2 cells.JPYSF treatment could not only improve renal function and pathological injury but also inhibit renal fibrosis in CKD mice. Additionally, JPYSF reversed fibrotic response in TGF-β1-induced HK-2 cells. Moreover, JPYSF rescued the decreased NAD+ content in CKD mice and TGF-β1-induced HK-2 cells through restoring expression of key enzymes in NAD+ biosynthesis, including quinolinate phosphoribosyltransferase, nicotinamide mononucleotide adenylyltransferase 1, and nicotinamide riboside kinase 1.JPYSF alleviated renal fibrosis in CKD mice and reversed fibrotic response in TGF-β1-induced HK-2 cells, which may be related to the restoration of NAD+ biosynthesis.