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Abstract Background EVs released by adipose derived stem cells (ADSCs) have shown promise as a therapeutic for tissue repair and regeneration because of their purported immune-regulatory properties. In this capacity, ADSC-EVs could be beneficial in improving graft retention rates for autologous fat grafting (AFG) post-mastectomy as, currently, grafted tissue rates are reported to be variable and low. Enriching grafted tissue with ADSC-EVs may improve retention rates by modulating macrophages resident within both the breast and lipoaspirate. We aimed to identify key macrophage phenotypes that are modulated by ADSC-EVs in vitro . Methods ADSCs were isolated from lipoaspirates of women undergoing AFG and characterised by flow cytometry and differentiation potential. ADSC-EVs were isolated from cell culture media and characterised by tunable resistive pulse sensing (TRPS), transmission electron microscopy (TEM), and Western blot. Primary monocyte-derived macrophages were polarized to an M1-like (GM-CSF, IFNγ) or M2-like phenotype (M-CSF, IL-4) or maintained (M0-like; M-CSF) and, at the time of polarization, ADSC-EVs were co-cultured with macrophages for 48 hrs. Flow cytometry coupled with high-dimensional analysis was used to cluster macrophages post co-culture. A manual gating strategy was generated to recapitulate these clusters and was applied to a repeat experimental run. Both runs were analysed to examine the prevalence of each cluster, representing a unique macrophage phenotype, with and without ADSC-EVs. Results Following the addition of ADSC-EVs, M0-like macrophages demonstrated a reciprocal shift of cell distribution from a cluster defined as having a ‘high inflammatory profile’ (CD36 +++ CD206 +++ CD86 +++ ; 38.6±14.8% of M1-like macrophages without ADSC-EVs; 16.5±7.0% with ADSC-EVs; p<0.0001) to a cluster with a ‘lower inflammatory’ profile (CD36 + CD206 + CD86 + ; 16.6±11.2% to 35±21.5%; p<0.05). There was no shift in M2-like clusters following treatment with ADSC-EVs. Conclusions ADSC-EVs are complex regulators of macrophage phenotype that can shift macrophages away from a heightened pro-inflammatory state.