Background: PM54 is a lurbinectedin analog that binds CG-rich promoter regions, inducing transcriptional blockade, DNA double-strand breaks, and S-phase arrest culminating in apoptosis. In gastric cancer models, it drives early transcriptional reprogramming with repression of cell-cycle and DNA repair pathways and demonstrates potent single-agent antitumor efficacy in vivo. We aimed to evaluate the antitumor efficacy of PM54 in gastric cancer and to assess its potential synergy with standard chemotherapeutic agents Methods: WNT/beta-catenin activity was assessed in gastric cancer cell lines using TCF/LEF reporter assays and pathway biomarker analysis. Drug interactions with 5-fluorouracil (5-FU) and cisplatin were quantified by combination index analysis. In vivo efficacy was tested in gastric cancer xenografts treated with PM54, chemotherapy, or combinations, with tumor growth monitored over time. Results: As a single agent, PM54 markedly inhibited the WNT/beta-catenin pathway, reducing TCF/beta-catenin-driven transcription by about 40% at 6 hours and over 70% at 18 hours post-treatment. In vitro, PM54 showed strong synergy with 5-fluorouracil (5-FU) in diffuse-type gastric cancer cell lines (HGC-27 and Hs746T) and with cisplatin in HGC-27 cells. In vivo, treatment with PM54 (0.9 mg/kg; days 0 and 7) of mice bearing HGC-27 xenograft tumors resulted in strong antitumor activity. On day 10 (last day of survival in the placebo-treated group), the median tumor volume in placebo-treated mice was 1612 mm³, compared with mice treated with 5-FU (1375 mm³), cisplatin (1423 mm³), and PM54 (616 mm³; p = 0.019). The combination of PM54 with 5-FU or cisplatin resulted in clearly superior performance to the corresponding therapies, with a median ΔT/ΔC (%) of 29.4 (PM54), 84.2 (5-FU), and 15.0 (combination); a similar pattern was observed with cisplatin, with ΔT/ΔC (%) 29.4 (PM54), 85.7 (cisplatin), and 21.5 (combination). Conclusions: PM54 represses WNT/beta-catenin signaling and enhances the efficacy of chemotherapy in gastric cancer models. These findings support its development in rational combination regimens to improve patient outcomes.