AbstractQuercetin is a plant flavonoid that has been recognized to have anti‐inflammatory, antioxidant and anti‐proliferative activities. This study aims to evaluate the inhibitory effects of quercetin against prostate malignancy in vitro and the underlying resistance mechanism. IC50values of quercetin were determined by MTT assay. Annexin‐V/PI staining was used to measure the rate of apoptosis. DNA cell cycle was analysed by PI staining method. Real‐time PCR was performed to assess mRNA levels ofOPNisoforms,VEGFisoforms,P53andKLK2. Migration potential, proliferative capability and nucleus morphology of cells were evaluated by the scratch‐wound assay, colony‐forming assay and Hoechst staining, respectively. Quercetin significantly increased the apoptosis rate of PC‐3 and LNCaP cell lines, arrested the cell cycle at the sub‐G1/G1 phase, and reduced the migration potential and colony‐forming capability. Moreover, upregulation of apoptosis‐related genes and downregulation of genes involved in proliferation and angiogenesis was also observed. Although our results elucidated that quercetin has antitumor effects on PC‐3 and LNCaP, for the first time, we showed that quercetin treatment causes alterations in the expression ofOPNandVEGFisoforms, which are cancer‐promoting modulators through various processes such as angiogenesis and drug‐resistance. Prostate malignant cells can dodge the anti‐carcinogenic properties of quercetin via modulation ofOPNandVEGFisoforms in vitro. Therefore, quercetin acts as a double‐edged sword in prostate cancer treatment.