Background <p>The inherently immune-cold microenvironment of prostate cancer (PCa) is a major barrier to the efficacy of immune checkpoint inhibitors (ICIs). Strategies capable of converting “cold” tumors into “hot” ones are therefore urgently needed. This study investigated how the traditional Chinese medicine prescription Fuzheng Yi’ai Formula (FZYAF) reverses the immunosuppressive state of PCa by inducing immunogenic cell death (ICD) and elucidated the underlying molecular mechanisms.</p> Methods <p>A combined strategy integrating bioinformatics, in vitro, and in vivo experiments was applied. Core PCa-related targets were first identified using weighted gene co-expression network analysis (WGCNA) and two-sample Mendelian randomization (MR). Ultra-performance liquid chromatography–mass spectrometry (UPLC-MS) was used to characterize active compounds in FZYAF and predict their potential targets. The effects of FZYAF-medicated serum on PCa cell proliferation, migration, invasion, and ICD hallmarks (cell-surface calreticulin (CRT) exposure and extracellular ATP and high-mobility group box 1 (HMGB1) release) were evaluated in vitro. Immunogenicity was assessed in vivo using a prophylactic tumor vaccine model in C57BL/6 mice. The involvement of key signaling pathways was further examined using the PERK-specific inhibitor GSK2606414, along with systematic analysis of the tumor immune microenvironment.</p> Results <p>Bioinformatics analysis identified ATF4, HMGB1, CYP1B1, and DLL1 as key causal targets linking FZYAF with PCa. In vitro experiments confirmed that FZYAF significantly inhibited PCa cell proliferation and motility in a dose-dependent manner, upregulated the expression of ATF4 and HMGB1, and effectively induced ICD hallmarks (CRT exposure, extracellular secretion of ATP and HMGB1). In vivo, FZYAF-prepared vaccines elicited protective anti-tumor immunity, an effect significantly attenuated by the PERK inhibitor GSK2606414. Furthermore, FZYAF treatment promoted the maturation of intratumoral dendritic cells and increased the infiltration of IFN-γ-secreting CD8⁺ T cells. Western blot analysis verified that FZYAF activated the PERK-eIF2α-ATF4-CHOP signaling pathway in vivo, and this activation was reversible by the PERK inhibitor.</p> Conclusion <p>FZYAF effectively induces immunogenic cell death in prostate cancer cells by activating the PERK-eIF2α-ATF4-CHOP signaling pathway. This study unveils a novel mechanism of FZYAF’s anti-tumor activity, establishing its potential as an ICD inducer and providing a solid theoretical basis for its future clinical application.</p>