<p>Nanomaterial-based ferroptosis inducers hold potential to overcome limitations of conventional cancer therapies, though most rely on glutathione peroxidase 4 (GPX4) downregulation. However, downregulation of GPX4 can impair the tumor immune system by inducing ferroptosis in tumor-associated neutrophils, potentially promoting tumor progression. Therefore, it is essential to explore GPX4-independent ferroptosis mechanisms specifically targeting tumor cells. Here, we demonstrate that the FDA-approved antidiabetic drug metformin potently enhances ferroptosis induced by ultrasmall manganese ferrite (MnFe₂O₄, MFO) nanoparticles in prostate cancer. Mechanistically, this combined effect operates independently of GPX4 suppression and hinges on metformin-mediated upregulation of transferrin receptor 1 (TfR1), which regulates iron homeostasis and elevates intracellular Fe²⁺ levels. Additionally, metformin promotes lipid peroxidation by upregulating acyl-CoA synthetase long-chain family member 4 (ACSL4). Notably, the combinational effect occurs only when prostate cancer cells first engulf sufficient MFO before metformin administration; simultaneous administration inhibits MFO uptake. To improve tumor-specific delivery, prostate-specific membrane antigen (PSMA) antibody-modified microbubbles assisted by ultrasound irradiation were used to facilitate precise intratumor accumulation of MFO in vivo. This therapeutic approach activates ferroptosis-mediated tumor suppression without GPX4 downregulation, underscoring the potential of sequential nanotherapy and metabolic regulation to synergistically overcome treatment resistance.</p> Graphical abstract <p></p>