Thymoquinone, a major ingredient of black seed oil (Nigella sativa), has been shown to exhibit anticancer capacity in various types of cancers. However, there are few studies concerning the correlation between thymoquinone and epithelial-to-mesenchymal transition (EMT) in prostate cancer. In the present study, we firstly found that thymoquinone showed antimetastatic capacity in prostate cancer DU145 and PC3 cells. Additionally, thymoquinone reversed EMT by increasing E-cadherin expression and decreasing vimentin and Slug expression in a concentration-dependent manner. Recent studies have shown that the transforming growth factor-β (TGF-β) signaling pathway may be associated with EMT. Intriguingly, the expression of TGF-β, Smad2 and Smad3 at the mRNA and protein levels was notably reduced upon thymoquinone treatment in prostate cancer DU145 and PC3 cells. Subsequently, we confirmed that thymoquinone repressed metastasis and EMT of prostate cancer through downregulation of the TGF-β/Smad2/3 signaling pathway, which may be partially reversed by TGF-β overexpression. In summary, our findings demonstrated that thymoquinone suppressed the metastatic phenotype and reversed EMT of prostate cancer cells by negatively regulating the TGF-β/Smad2/3 signaling pathway. These findings suggest that thymoquinone is a potential therapeutic agent against prostate cancer which functions by targeting TGF-β.