Heart transplantation is a crucial intervention for severe heart failure, yet the challenge of organ rejection is significant. Bone marrow mesenchymal stem cells (BMSCs) and their exosomes have demonstrated potential in modulating T cells, dendtitic cells (DCs), and cytokines to achieve immunomodulatory effects. DCs, as key antigen-presenting cells, play a critical role in shaping immune responses by influencing T-cell activation and cytokine production. Through this modulation, BMSCs and their exosomes enhance graft tolerance and prolonging survival.To explore the immunomodulatory effects of exosomes derived from BMSCs overexpressing microRNA-540-3p (miR-540-3p) on cardiac allograft tolerance, focusing on how these exosomes modulating DCs and T cells activity through the CD74/nuclear factor-kappaB (NF-κB) pathway.Rat models were used to assess the impact of miR-540-3p-enhanced exosomes on immune tolerance in cardiac allografts. MiR-540-3p expression was manipulated in BMSCs, and derived exosomes were collected and administered to the rat models post-heart transplantation. The study monitored expression levels of major histocompatibility complex II, CD80, CD86, and CD274 in DCs, and quantified CD4+ and CD8+ T cells, T regulatory cells, and cytokine profiles.Exosomes from miR-540-3p-overexpressing BMSCs lead to reduced expression of immune activation markers CD74 and NF-κB p65 in DCs and T cells. Rats treated with these exosomes showed decreased inflammation and improved cardiac function, indicated by lower levels of pro-inflammatory cytokines (interleukin-1β, interferon-γ) and higher levels of anti-inflammatory cytokines (interleukin-10, transforming growth factor β1). Additionally, miR-540-3p skewed the profiles of DCs and T cells towards immune tolerance, increasing the ratio of T regulatory cells and shifting cytokine secretion to favor graft acceptance.Exosomes derived from BMSCs overexpressing miR-540-3p significantly enhance immune tolerance and prolong cardiac allograft survival by modulating the CD74/NF-κB pathway, which regulates activities of DCs and T cells. These findings highlight a promising therapeutic strategy to improve heart transplantation outcomes and potentially reduce the need for prolonged immunosuppression.©The Author(s) 2024. Published by Baishideng Publishing Group Inc. All rights reserved.