Chemoresistance remains one of the major obstacles to cancer treatment. The search for specific molecules that could improve cancer treatment has become one of the objectives of biomedical research. Identifying new natural molecules to enhance chemotherapy treatment or improve sensitization to conventional therapies has become a key objective. Here, we evaluated the effect of Xanthohumol (XN) extracted from hop on SKMEL-28 melanoma cells and their sensitization to vemurafenib (VEM) treatment. We measured the XN effect on cell viability and apoptosis. We also assessed the effect of XN on membrane fluidity and membrane cholesterol levels. Finally, we studied the impact of XN on cell sensitization to VEM. Here, we showed that XN reduced SKMEL-28 cell viability through an apoptotic mechanism. Our results demonstrated the potential role of XN in sensitizing cancer cells to VEM with a less toxic effect on non-tumor cells. A study of XN's molecular mechanism showed that XN was able to induce cholesterol depletion and increased fluidity in SKMEL-28 cancer cells. This leads to an increase in VEM incorporation. Here, we describe the importance of the strategy to modulate membrane fluidity by XN in order to significantly improve anticancer therapy.