Objective To investigate the effect and mechanism of gentiopicroside (GPS) on renal interstitial fibrosis in unilateral ureteral obstruction (UUO) mice. Methods Twenty-five C57BL/6 male mice were randomly divided into the sham operation group, model control group, low-dose GPS group, high-dose GPS group, and irbesartan group, with 5 mice in each group. Mice in the sham operation group were fed normally, while in the other four groups, we established the UUO models by ligating one side of the ureter in mouse. After successful modeling, mice in the low-dose and high-dose GPS groups were given 0. 1 and 0. 2 g/kg GPS through intraperitoneal injection, mice in the irbesartan group were given 0. 02 g/kg irbesartan intraperitoneally, and mice in the model group and sham operation group were given equal volume of normal saline through intraperitoneal injection for 7 consecutive days. The mice were executed to take the kidney tissues to observe the morphological changes of kidney tissues using HE staining, and to observe the fibrosis of kidney tissues using Masson staining. Western blotting and RT-qPCR were used to detect the protein and mRNA expression levels of fibronectin (Fn) and α-smooth muscle actin (α-SMA) in the renal tissues. Another six mice were randomly divided into the model group and intervention group, with 3 mice in each. After the UUO models were established, the intervention group was given 0. 2 g/kg GPS through intraperitoneal injection, while the model group was given equal volume of normal saline intraperitoneally for 7 consecutive days. Mice were euthanized, the kidney tissues were taken for transcriptome sequencing, two sets of differentially expressed genes were analyzed, and GO and KEGG pathways enrichment were conducted. Finally, RT-qPCR was used for validation. Results Compared with the sham operation group, the model control group showed significant dilation of renal tubules, a large number of inflammatory cells in the renal tissues, and deposition of a large amount of collagen fibers in the renal interstitium. Compared with the model control group, the low-dose and high-dose GPS groups showed a certain degree of reduction in the renal tubular dilation and inflammatory cell infiltration in the kidney tissues, and improved renal interstitial fibrosis, with more significant effect in the high-dose GPS group. Compared with the sham operation group, the Fn,α-SMA protein and mRNA expression levels increased in the renal tissues of the model control group. Compared with the model control group, the Fn,α-SMA protein and mRNA expression levels decreased in the renal tissues of the low-dose and high-dose GPS groups (all P<0. 05), and those in the high-dose group were lower than those of the low-dose group. The sequencing analysis results showed that there were 710 differentially expressed genes between the model group and the intervention group. GO enrichment analysis showed that differentially expressed genes were mainly concentrated in cell processes, biological regulation, and biological process regulation; KEGG pathway enrichment analysis revealed that differentially expressed genes involved Ras, Rap1, PI3K/AKT, MAPK signaling pathways, etc. Two effective genes HGF and CCDC3 were screened for validation. Compared with the model group, the expression of HGF and CCDC3 in the intervention group increased (both P<0. 05), which was consistent with the sequencing results. Conclusion GPS can effectively alleviate renal fibrosis in UUO mice, and its mechanism of action may be related to up-regulating the expression of HGF and CCDC3, Rap1, Ras, PI3K/AKT, MAPK signaling pathways, and other biological pathways. [ABSTRACT FROM AUTHOR]