Rationale: Glioblastoma (GBM) is the most fatal form of brain cancer and its treatment represents a persistent challenge. Mesenchymal stromal cells (MSCs) have been explored as therapeutic tools in cancer management owing to their tumor-homing abilities. However, their clinical application is limited due to the controversial role of MSCs in carcinogenesis. This study investigates how MSCs influence tumor behavior and explores the synergistic anticancer effects in combination with melatonin (Mel). Methods: Orthotopic and subcutaneous GBM xenograft mouse models were used to assess the antitumor effect of Mel pre-treated MSCs (MSCMel). Histological, immunohistochemical, and ultrastructural analyses were conducted to identify phenotypic changes in tumors. Through a set of in vitro assays, including direct and indirect co-cultures, dynamic single-cell tracking and tumorsphere assay, we explored the impact of MSCMel on primary and non-primary GBM cells. Transcriptomic profiling was used to identify genes and pathways modulated by this synergistic therapy. Results: MSCMel delayed tumor growth in mice and increased collagen deposition. Additionally, MSCMel showed enhanced capacity to prevent GBM cell migration compared to untreated MSCs. Molecular analysis identified genes and proteins related to cell migration, cytoskeletal dynamics and extracellular matrix remodeling in GBM cells exposed to MSCMel, including reduced vimentin expression. Finally, a genetic signature associated with the clinical outcomes of GBM patients was identified. Conclusions: Our study demonstrates that melatonin enhances the anticancer properties of MSCs, providing new insights into their interaction with GBM cells and tumor environment. These findings offer valuable guidance for advancing MSC-based therapies in clinical practice.