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Background: As a member of the DExD/H-box RNA helicase family, DHX34 has demonstrated a significant correlation with the development of multiple disorders. Nevertheless, a comprehensive investigation between DHX34 and pan-cancer remains unexplored. Methods: We analyzed the value of DHX34 in pan-cancer based on some databases, such as The Cancer Genome Atlas (TCGA), Gene Expression Omnibus (GEO), and The Human Protein Atlas (HPA) by use the R language as well as some online analysis tools, including STRING, TISIDB, TISCH2. And based on our samples we performed Western blot (WB), qPCR and immunohistochemical staining (IHC) experiments. Results: DHX34 was highly expressed in most tumors, including Liver Hepatocellular Carcinoma (LIHC), compared to corresponding normal tissues. Among cervical cancers, DHX34 mutation frequency was the highest. Intriguingly, a positive correlation was observed between DHX34 expression and Mutational Burden (TMB) across 12 tumor types, and Microsatellite Instability (MSI) across 10 tumor types. Remarkably, DHX34 exhibited a favorable diagnostic value in a multitude of tumors. High expression of DHX34 is associated with poor prognosis in tumors such as adrenocortical carcinoma (ACC), renal papillary cell carcinoma (KIRP), low-grade glioma (LGG), and LIHC. Correlation analysis indicated that DHX34 expression correlated with clinicopathological features in a variety of tumors. The Protein-Protein Interaction (PPI) network and GSCALite database suggested that DHX34 and its ten co-expression genes might promote cancer progression by regulating the cell cycle. Gene Set Enrichment Analysis (GSEA) results further showed that DHX34 was positively correlated with pathways such as cell cycle, mitosis, and gene transcription regulation. The TISIDB database showed that DHX34 expression was closely associated with immune infiltration. Based on the TISCH2 database, we found that DHX34 was expressed in a number of immune cells, with relatively high expression in monocyte macrophages in LIHC. Conclusions: In summary, our study found that DHX34 is highly expressed in pan-cancer and has diagnostic and prognostic value. Targeting DHX34 may improve the therapeutic efficacy of immunotherapy and chemotherapy in a multitude of tumors., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).) more...