Glioblastoma are incurable primary tumors of the central nervous system that frequently harbor molecular alterations in the retinoblastoma pathway with subsequent cell cycle abnormalities. It is aimed to investigate the anti-glioma activity of the novel cell cycle-stabilizing compound Argyrin F and its potential treatment-induced vulnerabilities to exploit possibilities for novel rational combination therapies. Human and murine glioma cells are used, cytotoxicity and clonogenic survival assays, cell cycle analyses, and immunoblots are performed. Residual freshly resected tissue is used for a preclinical glioma model ex vivo, i.e., patient-derived microtumors (PDMs) for co-culture experiments with autologous tumor-infiltrating lymphocytes (TILs). Mass spectrometry-based immunopeptidomics are performed and the orthotopic syngeneic SMA560/VM/Dk glioma mouse model is used. Argyrin F-induced cell cycle arrest and reduced clonogenic survival is observed. Argyrin F-treated experimental glioma in vivo displays 4.6-fold more glioma-infiltrating CD8+ T cells. A distinctive treatment-induced immunopeptidome is discovered. The combination of Argyrin F plus PD-1 antibody increases cellular toxicity in PDM/TILs co-cultures ex vivo and prolonged overall survival compared with monotherapies in vivo. Thus, Argyrin F leads to anti-glioma effects and increases the immunogenicity, paving the way for a novel combination therapy of Argyrin F plus PD-1 blockade.