Autosomal dominant polycystic kidney disease (ADPKD) is the most common monogenetic inherited kidney disease characterized by renal progressive fluid-filled cysts and interstitial fibrosis. Inhibiting renal cyst development and interstitial fibrosis has been proven effective in delaying the progression of ADPKD. The purpose of this study was to discover effective drugs from natural products for preventing and treating ADPKD. Candidate compounds were screened from a natural product library by virtual screening. The Madin-Darby canine kidney (MDCK) cyst model, embryonic kidney cyst model, and orthologous mouse model of ADPKD were utilized to determine the pharmacological activities of the candidate compounds. Western blot and morphological analysis were used to investigate underlying mechanisms. The experimental results showed that 0.625, 2.5, and 10 μM cardamonin dose-dependently reduced formation and enlargement in MDCK cyst model. Cardamonin also significantly attenuated renal cyst enlargement in ex vivo mouse embryonic kidneys and PKD mouse kidneys. We found that cardamonin inhibited renal cyst development and interstitial fibrosis by downregulating the MAPK, Wnt, mTOR, and transforming growth factor-β/Smad2/3 signaling pathways. Cardamonin significantly inhibits renal cyst development and interstitial fibrosis, suggesting that cardamonin shows promise as a potential therapeutic drug for preventing and treating ADPKD.