CISD1, an outer mitochondrial membrane iron-sulfur cluster protein, regulates intracellular iron levels, oxidative stress, and mitochondrial dynamics, playing critical roles in cellular bioenergetics and redox homeostasis. Although CISD1 has been identified as a prognostic biomarker in specific cancers, its broader implications in tumorigenesis, cancer progression, and immunotherapy remain unclear. Given the heterogeneity of cancer and the need for robust biomarkers across cancers, this study conducts the first comprehensive pan-cancer analysis of CISD1 by evaluating its roles in cancer and treatment. We obtained and analyzed data from databases including TCGA, GTEx, THPA, GEPIA2.0, SangerBox, cBioPortal, TIMER2.0, CAMOIP, DAVID, SRPLOT, and TISIDB. Our findings reveal significant alterations in CISD1 expression at both transcriptional and translational levels, as well as gene mutations across multiple cancers, indicating its potential as a diagnostic biomarker and its involvement in cancer development and progression. CISD1 dysregulation is linked to poor clinical outcomes, as shown through its impact on patient prognosis. GO and KEGG analyses show that CISD1 plays critical roles in cellular bioenergetics. Notably, CISD1 expression is significantly correlated with tumor stemness indices, tumor mutation burden, microsatellite instability, and immune checkpoint proteins in multiple cancers, and altered CISD1 levels are also observed in patients responding to immunotherapy, further supporting its role not only in prognosis but also as a key predictor in immunotherapy responses and outcomes. Our findings demonstrate CISD1 as a reliable and promising diagnostic, prognostic, and immunotherapeutic biomarker for multiple cancers, emphasizing its crucial role in cancer biology and potential to guide personalized cancer therapies.