Abstract: Gastric cancer stem cells (GCSCs) contribute substantially to the refractory features of gastric cancer (GC) and are responsible for metastasis, relapse and drug resistance. However, strategies aimed at eliminating GCSCs to improve the clinical outcome of GC patients remain a challenge. Here, we report that tipranavir, a clinically used anti-HIV drug, effectively killed both GCSC and GC cell lines, and inhibited tumor growth in GCSC-derived xenograft models without apparent toxicity. Mechanistic analyses demonstrated that tipranavir induced GCSC cell apoptosis by targeting the novel serine protease PRSS23, releasing MKK3 from the PRSS23/MKK3 complex to activate p38 MAPK, and thereby activating the interleukin (IL) 24-mediated Bax/Bak mitochondrial apoptotic pathway. Tipranavir also killed other types of cancer cell lines and drug-resistant cell lines. Our findings indicate that by targeting both GCSC and GC cells, tipranavir is a promising anti-cancer drug, and the clinical development of tipranavir or other drugs specifically targeting the PRSS23/MKK3/p38MAPK–IL24 mitochondrial apoptotic pathway may offer an effective approach to combat gastric and other cancers.