Adiposity and diabetes affect breast cancer (BC) progression. We addressed whether glucose may affect the interaction between mammary adipose tissue-derived mesenchymal stromal/stem cells (MAT-MSCs) and BC cells. Two-dimensional co-cultures and spheroids were established in 25 mM or 5.5 mM glucose (High Glucose-HG or Low Glucose-LG) by using MAT-MSCs and MCF7 or MDA-MB231 BC cells. Gene expression was measured by qPCR, while protein levels were measured by cytofluorimetry and ELISA. CD44^high/CD24^low BC stem-like sub-population was quantified by cytofluorimetry. An in vivo zebrafish model was assessed by injecting spheroid-derived labeled cells. MAT-MSCs co-cultured with BC cells showed an inflammatory/senescent phenotype with increased abundance of IL-6, IL-8, VEGF and p16^INK4a, accompanied by altered levels of CDKN2A and LMNB1. BC cells reduced multipotency and increased fibrotic features modulating OCT4, SOX2, NANOG, αSMA and FAP in MAT-MSCs. Of note, these co-culture-mediated changes in MAT-MSCs were partially reverted in LG. Only in HG, MAT-MSCs increased CD44^high/CD24^low MCF7 sub-population and promoted their ability to form mammospheres. Injection in zebrafish embryos of HG spheroid-derived MCF7 and MAT-MSCs was followed by a significant cellular migration and caudal dissemination. Thus, MAT-MSCs enhance the aggressiveness of BC cells in a HG environment.