The extracellular matrix (ECM) is biochemically and biomechanically important for the structure and function of the mammary gland, which undergoes vast structural changes throughout pubertal and reproductive development. Although hyaluronan (HA) is a ubiquitous glycosaminoglycan (GAG) of the mammary gland ECM, extensive characterization of HA deposition in the mammary gland is lacking. Understanding physiologic HA metabolism is critical as this tightly controlled system is often hijacked in cancer. In the current studies, we characterize HA regulation throughout mammary gland development to better understand subsequent dysregulation of HA in mammary tumors. Using immunofluorescence (IF) imaging, we demonstrate that organized HA-rich septa exist in the mammary gland stroma throughout puberty, pregnancy, and involution. Furthermore, we find heterogeneous HA deposition within two murine models of breast cancer. Using cell specific isolation techniques, we characterize expression of genes associated with HA binding, synthesis, and degradation within EpCAM + epithelial cells, CD90.2 + fibroblasts, and F4/80 + macrophages isolated from mammary glands and tumors. Most notably, we identify elevated levels of the hyaluronidases Hyal1 and Hyal2 in tumor-association macrophages (TAMs), suggesting a role for TAM-mediated turnover of HA in the tumor microenvironment (TME). Gene expression is supported functionally by in vitro experiments in which macrophages treated with tumor-cell conditioned media exhibit increased hyaluronidase activity. These findings link TAMs to the direct degradation of HA within the TME of mammary tumors, which has negative implications for patient survival.