To study the therapeutic effect and mechanism of Pyrrosia petiolosa (P. petiolosa) extract on ethylene glycol- (EG-) induced urolithiasis in rats. Thirty SD male rats were randomly divided into five groups (n = 6): control group, EG group, and P. petiolosa group (25 mg/kg, 50 mg/kg group, and 100 mg/kg). Biochemical testing was adopted for measuring the blood and urine parameters, as well as the level of superoxide dismutase (SOD), glutathione (GSH), and malondialdehyde acid (MDA) in kidney tissues. HE staining and ELISA were utilized to observe the histopathological changes and detect the level of IL-1β, IL-6, MCP-1, and TNF-α in the kidney tissue, respectively. And western blot was performed for checking NOX2, NOX4, TGF-β1, p-Smad3, Smad3, p-Smad2, and Smad2 protein expression level in kidney tissues. EG could significantly increase the level of blood urea nitrogen, creatinine, and Na in serum and 24-hour urinary protein, oxalate, uric acid, creatinine, calcium, and phosphorus in urine and decreased the urine volume in rats. Whereas P. petiolosa extract was able to greatly decrease the level of related parameters in serum and urine in a dose-dependent manner, but did not affect the urine pH. In addition, P. petiolosa extract notably ameliorated EG-induced renal tissue injury. Compared with the EG group, P. petiolosa extract markedly raised the level of SOD and GSH and decreased the MDA level and the expression of NOX2 and NOX4 in the kidney tissue. Moreover, P. petiolosa extract also lowered the level of IL-1β, IL-6, MCP-1, and TNF-α in EG-stimulated kidney tissue and inhibited the protein level of EG-induced TGF-β1, p-Smad3, and p-Smad2 in a concentration-dependent manner. P. petiolosa extract can improve EG-induced urolithiasis in rats by inhibiting oxidative stress, inflammatory response, and the activation of TGF-β pathway.