Objective <p>Cancer cells derive the majority of their energy necessary for rapid proliferation and growth through glycolysis, irrespective of oxygen availability and mitochondrial functionality. Mitochondrial transfer has been explored as an innovative therapeutic approach for various diseases; however, there are divergent perspectives on whether mitochondrial dysfunction—frequently observed in cancer cells—is a cause or a consequence of tumor progression. 3-BP inhibits hexokinase, thereby blocking the entry step of glycolysis. This study aims to investigate the effects of 3-Bromopyruvic acid (3-BP), a hexokinase inhibitor, in conjunction with mitochondrial transfer on DU-145 prostate cancer cells.</p> Materials and methods <p>In this study, human bone marrow-derived mesenchymal stem cells (HMSCs) were utilized as donors for isolated mitochondria intended for mitochondrial transfer. ATP levels of the isolated mitochondria were quantified. The mitochondria were labeled with Mitotracker Red CMXRos or MitoSpy Green, and fluorescence microscopy was used to assess co-localization of donor-derived mitochondrial signal with recipient cells, interpreted as evidence compatible with cellular association/uptake rather than proof of stable integration. Mitochondrial transfer was conducted by co-incubating the mitochondria with DU-145 cells with the addition of various doses of 3-BP for 24 and 48&#xa0;h. The sensitivity of the cells to 3-BP and cell proliferation post-mitochondrial transfer were evaluated using the MTT assay (3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide). Apoptosis and in situ cell death were assessed employing the TUNEL method. Caspase-3 levels were measured via ELISA, while mTOR and HIF1α protein expressions were determined through western blot analysis.</p> Results <p>The antiproliferative efficacy of 3-BP alone and in conjunction with mitochondrial transfer was evaluated in DU-145 cells. Mitochondria isolated from HMSCs exhibited viability and functionality, maintained adequate ATP levels, and showed donor-derived signal detectable inside recipient cells alongside recipient mitochondrial staining in DU-145 cells. While 3-BP application significantly inhibited the proliferation of DU-145 cells, mitochondrial transfer alone did not show a significant antiproliferative effect. However, the sequential application of both treatments effectively suppressed cell growth. Sequential treatment resulted in a reduction of overall mTOR and HIF-1α protein levels as determined by Western blot analysis under the specified experimental conditions. These measurements denote relative expression levels; however, they do not, in isolation, confirm the activity of the mTOR pathway. Caspase-3 activity and TUNEL positivity remained unaltered across different groups, which aligns with the assay-specific detection of distinct apoptotic features observed at a single time point.</p> Conclusion <p>This study, in addition to being the first in vitro investigation of sequential mitochondrial transfer with 3-BP administration in prostate cancer cells, demonstrated that inhibiting glycolysis and mitochondrial transfer may serve as significant, alternative adjunctive approaches in cancer therapy.</p>