Sorafenib is the only chemotherapeutic agent currently approved for unresectable hepatocellular carcinoma (HCC). However, poor response rates have been widely reported. Indole-3-carbinol (I3C) is a potential chemopreventive phytochemical. The present study aimed to explore the potential chemomodulatory effects of I3C on sorafenib in HCC cells as well as the possible underlying mechanisms. I3C exhibited a greater cytotoxicity in HepG2 cells compared to Huh-7 cells (p < 0.0001). Moreover, the co-treatment of HepG2 cells with I3C and sorafenib was more effective (p = 0.002). Accordingly, subsequent mechanistic studies were carried on HepG2 cells. The results show that the ability of I3C to enhance sorafenib cytotoxicity in HCC cells could be partially attributed to increasing the apoptotic activity and decreasing the angiogenic potentials. The combination had a negative effect on epithelial-mesenchymal transition (EMT). Increased NOX-1 expression was also observed which may indicate the involvement of NOX-1 in I3C chemomodulatory effects. Additionally, the combination induced cell cycle arrest at the G0/G1 phase. In conclusion, these findings provide evidence that I3C enhances sorafenib anti-cancer activity in HCC cells.