Background 14 million people are diagnosed with new cancer and approximately 8 million people die from cancer every year. Hepatocellular carcinoma is the most common type of liver cancer and covers almost 5-6% of cancer deaths worldwide. Silybum marianum, a plant that contains silymarin, has been used traditionally in the treatment of liver diseases for centuries. The antioxidant, anti-inflammatory and anti-fibrotic anti-cancer properties of silymarin have been demonstrated in several studies in vivo and in vitro. The Slit/Robo signaling pathway plays a role in many processes such as neurogenesis, angiogenesis, cell proliferation, cell movement, cancer progression, cell invasion, migration and metastasis. In this study, we aimed to investigate the effects of silymarin on HepG2 Hepatocellular carcinoma cells on Slit-2/Robo-1 signaling pathway and CXCR-4 which plays a role in the metastasis process. Methods HepG2 Hepatocellular carcinoma cells were used in the study. Different doses of silymarin's effect on HepG2 cells were observed by hematoxylin and eosin staining. Immunoblotting techniques were used to test the expression of Slit-2/Robo-1 and CXCR4 protein level. Immunocytochemistry was used to visualize the localization of Slit-2/Robo-1 and CXCR4 protein within the cells. Results Silymarin caused apoptosis in HepG2 cells, decreased the level of CXCR-4 protein dose-dependently, and decreased the Slit-2/Robo-1 protein level at low doses and increased it at high doses. Conclusions Silymarin doses showed anti-carcinogenic, anti-metastatic and apoptotic effects in a dose-dependent manner on HepG2 cells through the Slit-2/Robo-1 pathway.