To investigate the role and molecular mechanism of estrogen deficiency in fluorine ion (F^–)-induced renal fibrosis, the models of F^– exposure in ovary removed rats were established by drinking water with different doses of F^– (0, 25, 50 and 100 mg/L) for 90 days. Results of H&E staining and BrdU labeled experiment showed that F^– induced renal pathomorphological damage and inhibited cell proliferation. Further, Masson staining showed that F^– induced renal glomerular and tubulointerstitial fibrosis. Meanwhile, renal fibrosis was confirmed by detecting the expression levels of collagen I, collagen III, collagen IV and fibronectin using immunofluorescence. In the state of estrogen deficiency, F^–-induced renal damage and fibrosis were aggravated. Moreover, the molecular mechanism of F^–-induced renal fibrosis was evaluated, and the results showed that F^– induced TGF-B1/Smad signaling pathway further dysregulation after ovariectomy, which manifested as the further up-regulated expression of TGF-B1, Smad2, p-Smad2, Smad3 and p-Smad3, and further down-regulated of Smad7. Accompanied by renal damage and renal fibrosis, renal function was also disturbed, especially in ovariectomized rats. This study indicated that estrogen deficiency aggravated F^–-induced renal fibrosis via the TGF-B1/Smad signaling pathway, leading to more serious renal dysfunction.