Background <p>The number of patients with radiation-induced premature ovarian failure (POF) has increased rapidly. In this research, we investigated the therapeutic effects and molecular mechanisms of the classic TCM prescription Zuogui Wan (ZGW) in radiation-induced POF.</p> Methods <p>SD rats and ovarian granulosa cells were exposed to γ-radiation with or without ZGW treatment. The morphological alterations in the ovaries, cell death, and mitochondrial damage were identified through H&amp;E staining, Hoechst/PI staining and TEM, respectively. Serum hormone levels were quantified using ELISA. Western blot was employed to assess the protein expression levels of GPX4 and DNMT3b. The RNA expression levels of miR-21 and DNMT3b were determined by PCR. Fe²⁺, MDA and GSH content were detected using the corresponding assay kit. FerroOrange was used for Fe²⁺ visualization.</p> Results <p>Radiation-induced POF and ferroptosis have been observed both in vivo and in vitro. ZGW strongly inhibited the increase in intracellular Fe²⁺ and MDA levels, as well as the decrease in estradiol, AMH, GSH, and GPX4 levels induced by radiation, exerting effects of anti-POF and anti-ferroptosis. miR-21-5p targeted DNMT3b and suppressed its expression. Inhibition of miR-21-5p or overexpression of DNMT3b counteracted the protective effect of ZGW.</p> Conclusion <p>ZGW alleviated radiation-induced POF and ferroptosis through the miR-21-5p/DNMT3b axis.</p>