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miR-101–3p may play a therapeutic role in various tumours. However, its anti-tumour mechanism remains unclear, and a definitive strategy to treat tumour cells in vivo is lacking. The objective of this study was to investigate the inhibitory mechanism of miR-101–3p on tumour cells and to develop relevant nanomedicines for in vivo therapy. The expression levels of miR-101–3p and its target protein TBLR1 in tumour tissues and cells were detected, and their relationship with ferroptosis was clarified. Furthermore, the efficacy of nanocarriers in achieving in vivo therapeutic gene delivery was evaluated. Nanomedicine was further developed, with the anti-proliferative in vivo therapeutic effect validated using a subcutaneous xenograft cancer model. The expression level of miR-101–3p negatively correlated with clinical tumour size and TNM stage. miR-101–3p restores ferroptosis in tumour cells by directly targeting TBLR1, which in turn promotes apoptosis and inhibits proliferation. We developed nanomedicine that can deliver miR-101–3p to tumour cells in vivo to achieve ferroptosis recovery, as well as to inhibit in vivo tumour proliferation. The miR-101–3p/TBLR1 axis plays an important role in tumour ferroptosis. Nanopharmaceuticals that increase miR-101–3p levels may be effective therapies to inhibit tumour proliferation.