Objective To observe expression of HIF-1α, RhoA and SOX9 proteins in kidneys and to determine their relationship with tubulointerstitial fibrosis in rats with adenine-induced chronic kidney disease. Methods Twenty rats were randomly divided into a control (Con) group and model group ( adenine-induced CKD rat model). The rats were sacrificed after 3 and 6 weeks. Morphological changes of the kidney were observed and the renal functions and 24 h urinary protein were assessed. Renal pathological changes were observed by HE staining and interstitial fibers of renal tubules were observed by Sirius red staining. Immunohistochemical staining was used to observe expression of HIF-1α, RhoA, SOX9, Col-I and α-SMA proteins in renal tissue. HIF-1α, RhoA, SOX9, Col-I and α-SMA mRNA levels in the kidney were measured by RT-PCR and the relationship between the expression levels of HIF-1α, RhoA and SOX9, and renal tubular interstitial fibrosis was analyzed. The expression level of SOX9 in serum of the two groups was measured by an ELISA. Results Compared with the Con group, 24 h urinary protein, serum BUN and Scr in the CKD group were increased significantly (P<0. 05). In the CKD group, HE staining and Sirius red staining showed obvious renal tubule dilatation, interstitial fibrosis and aggravation over time. Immunohistochemistry and RT-PCR showed that the expression of HIF-1 α, RhoA and SOX9 was increased in the CKD group and the serum level of SOX9 in the CKD group was higher than that in the control group, which was increased over time. Correlation analysis showed that the mRNA expression of HIF-1α, RhoA and SOX9 was correlated with Col-I and α-SMA, and there was a positive correlation among HIF-1α, RhoA and SOX9. Conclusions HIF-1α, RhoA and SOX9 are highly expressed in the adenine-induced CKD rat model, which may be closely related to the progression of renal interstitial fibrosis. Moreover, the serum level of SOX9 is expected to be a molecular marker for early diagnosis of CKD.