Objective <p>To explore the potential role and mechanism of platycodin D (PD) overcomes cisplatin (DDP) resistance in gastric cancer cells.</p> Methods <p>DDP-resistant gastric cancer cells AGS/DDP and HGC-27/DDP were constructed. The cells were divided into the following 6 groups, including erastin (10 µg/mL), PD (20 µmol/L), PD+ferrostatin-1 (Fer-1, 50 nmol/L), PD + overexpression negative control (OE-NC, 2 µg/µL), PD + overexpression of Tousled-like kinase 1 (OE-TLK1, 2 µg/µL), PD+ overexpression of DNA methyltransferase 3 alpha (OE-DNMT3A, 2 µg/µL). Cell viability and proliferation were assessed using cell counting kit-8 (CCK-8) or EdU kits. Ferroptosis-related indices including Fe²⁺, malondialdehyde (MDA) and reactive oxygen species (ROS) were measured employing relevant kits. The interactions between TLK1 and ubiquitin-like with PHD and ring finger domains 2 (UHRF2) or UHRF2 and DNMT3A were confirmed utilizing co-immunoprecipitation (Co-IP) assays. The ubiquitination of DNMT3A was analyzed through immunoprecipitation. <i>In vivo</i>, efficacy of PD was evaluated using subcutaneous graft tumors implanted in nude mice. DDP-resistant AGS gastric cancer cells (AGS/DDP, 1 × 10⁷ cells) were injected into the subcutaneous of nude mice, 1-week post-injection, DDP (5 mg/kg) was intraperitoneally injected 3 times per week. Male nude mice were randomly divided into 4 groups (<i>n</i>=6): control (0.9% saline), PD (10 mg/kg, intraperitoneally daily), PD + OE-NC (2 × 10¹⁰ vp/dose, subcutaneous injection) and PD + OE-TLK1 (2 × 10¹⁰ vp/dose, subcutaneous injection). Tumor volume growth curves were recorded and tumor weights were measured at the end of the experiment. Proliferation marker protein KI-67 and glutathione peroxidase 4 (GPX4) expressions were detected using immunohistochemical staining. Western blot analysis was performed to detect TLK1, phosphorylated-UHRF2 (p-UHRF2), UHRF2, DNMT3A, and arachidonate 15-lipoxygenase (ALOX15), ferroptosis-related proteins GPX4 and solute carrier family 7 member 11 (SLC7A11) expressions.</p> Results <p>Compared with the control group, PD treatment significantly inhibited cell proliferation of AGS/DDP, HGC-27/DDP cell lines (both <i>P</i>&lt;0.01) and induced ferroptosis in the DDP-resistant cell lines (<i>P</i>&lt;0.05 or <i>P</i>&lt;0.01). Furthermore, PD decreased the TLK1-mediated phosphorylation of UHRF2. TLK1 suppressed the ubiquitination degradation of DNMT3A via enhancing UHRF2 phosphorylation at Serine 643 site. Additionally, DNMT3A decreased the ALOX15 expression level via promotion of methylation in ALOX15 CpG island. <i>In vivo</i>, PD significantly reduced the growth of DDP-resistant gastric cancer cells (<i>P</i>&lt;0.01).</p> Conclusion <p>PD effectively restrained DDP resistance by promoting ferroptosis via targeting TLK1/UHRF2/DNMT3A/ALOX15 axis, which enriched the mechanism of PD in alleviation of cisplatin resistance in gastric cancer cells.</p>