Abstract: Objective To evaluate the effect of pirarubicin (THP) in combination with hyperthermia on gastric cancer tissues in vitro and explore the underlying mechanisms. Methods In vitro three-dimensional culture models were established with tissue biopsies from 36 patients with pathologically confirmed gastric cancer. The tumor cell viability was measured by MTS-PMS assay, and HE staining was used to study the histomorphological changes of the tissues following chemotherapy and hyperthermia. Results Synergistic tumor cell-killing effects of cisplatin, THP, and mitomycin with hyperthermia was observed in the tumor tissues (P=0.000), and THP exhibited stronger cytotoxic effects than the other drugs. Histomorphological study suggested strong killing effects of THP on the tumor tissues, which displayed disrupted tissue structure, cellular degradation and necrosis, karyopyknosis and karyolysis, with cytoplasm loss. The anti-tumor effects of THP were associated with clinical staging and pathological grading of the tumors (P=0.000), but not with the patients’ gender, age, tumor size and preoperative CEA levels (P>0.05). Conclusion Pirarubicin shows good synergistic effects with hyperthermia, and the cytotoxicity of pirarubicin against gastric cancer tissue is enhanced considerably by mild hyperthermia. THP can be a potential therapeutic drug for intraperitoneal chemohyperthermia.