BACKGROUND: The long-term effectiveness of immunotherapies against Multiple Myeloma (MM) remains elusive, demonstrated by the inevitable relapse in patients. This underscores the urgent need for an in-depth analysis of the MM tumor-immune microenvironment (TME). Hereto, a representative immunocompetent MM mouse model can offer a valuable approach to study the dynamic changes within the MM-TME and to uncover potential resistance mechanisms hampering effective and durable therapeutic strategies in MM. METHODS: We generated a comprehensive single-cell RNA-sequencing atlas of the MM-TME in bone marrow and spleen encompassing different stages of disease, using the immunocompetent 5T33MM mouse model. Through comparative analysis, we correlated our murine dataset with the pathogenesis in MM patients by reanalyzing publicly available datasets of human bone marrow samples across various disease stages. Using flow cytometry, we validated the dynamic changes upon disease progression in the 5T33MM model. Furthermore, interesting target populations, as well as the immune-boosting anti-CD40 agonist (αCD40) therapy were tested ex vivo on murine and human primary samples and in vivo using the 5T33MM model. RESULTS: In this study, we identified the heterogenous and dynamic changes within the TME of murine and human MM. We found that the MM-TME was characterized by an increase in T cells, accompanied with an exhausted phenotype. Although neutrophils appeared to be rather innocuous at early disease stages, they acquired a pro-tumorigenic phenotype during MM progression. Moreover, conventional dendritic cells (cDCs) showed a less activated phenotype in MM, underscoring the potential of immune-boosting therapies such as αCD40 therapy. Importantly, we provided the first pre-clinical evaluation of αCD40 therapy and demonstrated successful induction of cDC- and T-cell activation, accompanied by a significant short-term anti-tumor response. CONCLUSIONS: This resource provides a comprehensive and detailed immune atlas of the evolution in human and murine MM disease progression. Our findings can contribute to immune-based patient stratification and facilitate the development of novel and durable (immune) therapeutic strategies in MM.