Among other cancers, prostate cancer is globally the second most rampant one with the incidence of 29.4% among men. SLIT2/ROBO1 signaling is very crucial pathway causally implicated in many cancers and reported to inhibit a variety of cancer cell types. CXCR4 is a chemokine receptor implicated in cancer progression. Silymarin is a phytochemical, of which anti-carcinogenic activity was suggested in various cancers, including prostate cancer. However, there are no studies examining the effect of silymarin on SLIT2-Robo1-CXCR4 axis. Herein, our goal is to explore cytotoxic and morphological effects of silymarin on DU145 cells and to reveal its role in Slit2/Robo and CXCR1 pathway. First, 24, 48 and 72 h-long cytotoxicity tests were performed for dose analysis of silymarin, followed H-E stain for morphological evaluation with varying doses of silymarin. Afterward, western blot and immunocytochemistry analyses were carried out for SLIT2, ROBO1 and CXCR4 proteins. According to MTT analysis, IC50 concentrations for silymarin were 315, 126 and 70 mu M against DU145 cells for 24, 48 and 72 h treatments. In H-E, several apoptotic hallmarks, including, condensed, kidney-shaped and eccentric nuclei, membrane blebbings and apoptotic body formations were observed. Silymarin increased the expressions of SLIT2 and ROBO1 while decreased CXCR4 when compared to control group in immunocytochemistry and Western blot. To summarize, silymarin inhibited DU145 cells dose-dependently by activating SLIT2 protein and inhibiting expression of CXCR4. This study is the first examining the interplay between Slit2-Robo1-CXCR4 proteins and silymarin in DU145 cells. We believe that our study will provide new insights for future studies.