Objective Triple-negative breast cancer lacks effective targeted therapies, necessitating novel treatment approaches.To evaluate the antitumor efficacy and molecular mechanisms of l-rhamnose in TNBC. Methods We employed an integrated approach combining in vitro cell-based assays and in vivo xenograft models. TNBC cell lines (MDA-MB-231, Hs578T) were treated with l-rhamnose to assess proliferation and colony formation. Molecular docking predicted the binding between l-rhamnose and GSTM5. GSTM5 gain- and loss-of-function experiments were performed to confirm its functional role. The in vivo efficacy was evaluated using MDA-MB-231 nude mouse xenografts. Ferroptosis dependency was confirmed using Ferrostatin-1. Results l-rhamnose dose- and time-dependently inhibited TNBC proliferation and colony formation. Intraperitoneal administration (5 g/kg) significantly suppressed tumor growth without affecting mouse body weight. Fer-1 attenuated l-rhamnose-mediated tumor suppression both in vitro and in vivo. Mechanistically, l-rhamnose downregulated GSTM5 protein levels, concomitant with GPX4 reduction and lipid peroxide accumulation. GSTM5 overexpression rescued l-rhamnose-induced cell death and restored GPX4, whereas GSTM5 knockdown sensitized cells to ferroptosis. Molecular docking predicted a potential interaction between l-rhamnose and the GSTM5 protein, with a calculated binding energy of -5.341 kcal/mol, which guided our subsequent functional investigations. Conclusion l-rhamnose suppresses TNBC growth by inducing ferroptosis, a process associated with the downregulation of GSTM5. Our findings reveal that GSTM5 is a key mediator modulating ferroptosis sensitivity in TNBC. These results position l-rhamnose as a candidate for further investigation in TNBC therapy.