In recent years, cancer had an increase in recurrences, due to its heterogeneity and molecular complexity. This could explain, in part, the failure in the efficacy of treatment for cancer patients. Tumor microenvironment (TME) consists of cells and macromolecules that surround a tumor cell. Its main non-cellular component is the extracellular matrix (ECM), a complex network of proteins and polysaccharides that is highly deregulated in tumor context. Among the ECM components that are altered in tumors are hyaluronan (HA) and molecules associated with its metabolism. On the other hand, TME alterations can influence the expression of tumor-associated genes, such as BRCA1 and BRCA2, mainly involved in DNA repair. The aim of this study was to analyze alterations in HA signaling (CD44), synthesis (HAS2) and degradation (HYAL1) in tumor tissue (TT) and non-tumor adjacent tissue (NAT) from breast cancer patients, and its association with the expression of BRCA1 and BRCA2 and with patients’ clinical data. Additionally, we analyzed the expression of these genes in 3D spheroids of MDA-MB-231 and MCF-7 breast cancer cells after HA degradation (HYAL) or inhibition of its synthesis (4-MU), also evaluating the volume and area of the spheroids generated. mRNA expression in both patients and cell samples was evaluated by RT-qPCR. The levels of HA and BRCA1, BRCA2 and CD44 proteins from patients were analyzed by immunohistochemistry. Breast cancer patients showed lower levels of HAS2 (mRNA) and BRCA1 and BRCA2 (protein) in TT compared to NAT. Besides, we found a strong positive correlation between CD44 and HYAL1 mRNAs. In turn, a decrease in the size of the spheroids was observed when HA synthesis was inhibited. This work presents important evidence regarding the influence of ECM, particularly HA and molecules associated with its metabolism on DNA repair genes, which is crucial for identifying key markers to predict tumor progression and guide potential therapeutic approaches.