Tumor extracellular matrix (TECM) influence drug resistance due to an imbalance in the synthesis and degradation of its components, including hyaluronan (HA). HA accumulates in the TECM, impeding drug distribution and inducing pro-tumoral signals. UDP-glucuronic acid (UDP-GlcA) together with N-acetyl-glucosamine, are involved in HA synthesis. UDP-GlcA plays a key role in the elimination of chemotherapeutic drugs as epirubicin (EPI). On the other hand, candidate molecules for drug repositioning include 4-methylumbelliferone (4MU), an orally approved dietary supplement derived from coumarins. 4MU specifically inhibits HA synthesis by binding to UDP-GlcA and depleting the cellular pool required for HA synthesis. The aim was to propose a treatment with EPI + 4MU in two breast cancer models to reduce EPI elimination and affect HA synthesis. Spheroids of MDA-MB-231 and MCF-7 cells were established and after 5 days they were treated with EPI, 4MU, or EPI + 4MU for 3 days. EPI + 4MU treatment reduced tumor cell viability (MTS) and increased early apoptosis (flow cytometry) compared to control conditions. This effect was associated with a higher intracellular accumulation of EPI (flow cytometry), a decrease in spheroids size (microscopy) and the downregulation of efflux pumps involved in EPI resistance (RT-qPCR). Besides, EPI + 4MU treatment decreased the expression of the major HA synthase HAS2 and showed a tendency to increase hyaluronidases HYAL1 and HYAL2 (RT-qPCR). Moreover, this effect was shown when HA accumulation on the cell surface of tumor cells obtained from treated spheroids was determined by flow cytometry. In conclusion, 4MU promotes remodeling of the breast cancer ECM, affecting HA metabolism. Indeed, the combination of 4MU with EPI reduced EPI inactivation and elimination. Finally, combined treatment reduced the activation of cellular mechanisms involved in drug resistance, sensitizing tumor cells to EPI and enhancing the efficacy of antitumor therapy.