<jats:title>Abstract</jats:title><jats:sec> <jats:title>Background</jats:title> <jats:p>BCMA CAR-T is highly effective for relapsed/refractory multiple myeloma(R/R-MM) and significantly improves the survival of patients. However, the short remission time and high relapse rate of MM patients treated with BCMA CAR-T remain bottlenecks that limit long-term survival. The immune microenvironment of the bone marrow (BM) in R/R-MM may be responsible for this. The present study aims to present an in-depth analysis of resistant mechanisms and to explore potential novel therapeutic targets for relapse of BCMA CAR-T treatment via single-cell RNA sequencing (scRNA-seq) of BM plasma cells and immune cells.</jats:p> </jats:sec><jats:sec> <jats:title>Methods</jats:title> <jats:p>This study used 10X Genomic scRNA-seq to identify cell populations in R/R-MM CD45<jats:sup>+</jats:sup> BM cells before BCMA CAR-T treatment and relapse after BCMA CAR-T treatment. Cell Ranger pipeline and CellChat were used to perform detailed analysis.</jats:p> </jats:sec><jats:sec> <jats:title>Results</jats:title> <jats:p>We compared the heterogeneity of CD45<jats:sup>+</jats:sup> BM cells before BCMA CAR-T treatment and relapse after BCMA CAR-T treatment. We found that the proportion of monocytes/macrophages increased, while the percentage of T cells decreased at relapse after BCMA CAR-T treatment. We then reclustered and analyzed the alterations in plasma cells, T cells, NK cells, DCs, neutrophils, and monocytes/macrophages in the BM microenvironment before BCMA CAR-T treatment and relapse after BCMA CAR-T treatment. We show here that the percentage of BCMA positive plasma cells increased at relapse after BCMA CAR-T cell therapy. Other targets such as CD38, CD24, SLAMF7, CD138, and GPRC5D were also found to be expressed in plasma cells of the R/R-MM patient at relapse after BCMA CAR-T cell therapy. Furthermore, exhausted T cells, TIGIT<jats:sup>+</jats:sup>NK cells, interferon-responsive DCs, and interferon-responsive neutrophils, increased in the R/R-MM patient at relapse after BCMA CAR-T cell treatment. Significantly, the proportion of IL1β<jats:sup>hi</jats:sup> Mφ, S100A9<jats:sup>hi</jats:sup> Mφ, interferon-responsive Mφ, CD16<jats:sup>hi</jats:sup> Mφ, MARCO <jats:sup>hi</jats:sup> Mφ, and S100A11<jats:sup>hi</jats:sup> Mφ significantly increased in the R/R-MM patient at relapse after BCMA CAR-T cell therapy. Cell–cell communication analysis indicated that monocytes/macrophages, especially the MIF and APRIL signaling pathway are key players in R/R-MM patient at relapse after BCMA CAR-T cell therapy.</jats:p> </jats:sec><jats:sec> <jats:title>Conclusion</jats:title> <jats:p>Taken together, our data extend the understanding of intrinsic and extrinsic relapse of BCMA CAR-T treatment in R/R-MM patient and the potential mechanisms involved in the alterations of antigens and the induced immunosuppressive microenvironment, which may provide a basis for the optimization of BCMA CAR-T strategies. Further studies should be performed to confirm these findings.</jats:p> </jats:sec>