<p>Non-small cell lung cancer (NSCLC) is the most prevalent and lethal form of lung cancer, and it requires innovative therapies. Repurposing approved non-oncology drugs offers a promising strategy for developing novel and effective combination therapies. In the present study, we optimized and characterized the combination of an antifungal agent, posaconazole (PCZ), with arsenic trioxide (ATO), an anti-cancer drug whose clinical use is limited by toxicity and resistance, in A549 lung cancer cells. The cytotoxic effects of PCZ and ATO combination was analyzed through a comprehensive cellular investigation. Cell viability (IC₅₀), apoptosis, survival and proliferation were determined using MTT, fluorescence staining, flow cytometry, colony formation and cell cycle arrest assays. In addition, to explore the underlying molecular mechanisms western blot and qPCR assessments were employed to examine the roles of key survival and proliferation elements, Bcl-2 and HMGB1. The IC₅₀ value of ATO and PCZ as a single drug was 75 µM and 100 µM, respectively. In contrast, simultaneous combination of PCZ and ATO reduced IC₅₀ value of ATO to 25 µM. Replacement of ATO with doxorubicin (DOX), daunomycin (DNM) or berberine (BBR) represented that PCZ significantly enhances DOX toxicity but not DNM and BBR. The combination treatment also inhibited the colony forming capacity and increased cell cycle arrest at G2/M phase. Combination-induced apoptosis was mainly dominated by HMGB1 and Bcl-2 overexpression. PCZ synergistically and effectively reduces the adverse cytotoxic effects of ATO via HMGB1 and Bcl-2 signaling pathways, providing a new therapeutic strategy for lung cancer therapy.</p>