BACKGROUND Acute myeloid leukemia (AML) is a highly heterogeneous disease characterized by complex genetic and molecular features that contribute to poor prognosis and low cure rates. Therefore, identifying novel therapeutic targets is crucial for improving treatment efficacy and patient survival. This study investigated the potential role of cyclin-dependent kinase 9 (CDK9), a known regulator of gene expression, in AML pathogenesis and prognosis. METHODS This study employed multiple bioinformatics approaches, including analysis of CDK9 expression across various cancers using the Tumor Immune Estimation Resource (TIMER2.0) database and further investigation of its expression and prognostic significance in AML using data from the Cancer Genome Atlas (TCGA) and Genotype-Tissue Expression (GTEx) databases. Survival analysis and Cox regression analysis were used to assess the association between CDK9 expression and patient prognosis. Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and Gene Set Enrichment Analysis (GSEA) were performed to elucidate the pathways and biological processes influenced by CDK9. Furthermore, the relationship between CDK9 expression and tumor immune infiltration was evaluated, and a protein-protein interaction (PPI) network was constructed. In vitro experiments, including Western blotting, CCK-8 assays, and flow cytometry, were conducted to validate the bioinformatics findings. RESULTS Bioinformatics analysis revealed significantly elevated CDK9 expression in AML samples, which correlated with poor patient prognosis. Functional enrichment analysis indicated that CDK9 is involved in key pathways related to cell proliferation, differentiation, and the tumor microenvironment. Moreover, the study observed a strong correlation between CDK9 expression and altered immune cell infiltration, suggesting a potential role in immune evasion. In vitro experiments confirmed that CDK9 overexpression promoted AML cell proliferation and inhibited apoptosis. Additionally, CDK9 showed a strong correlation with epithelial-mesenchymal transition (EMT)-related proteins, suggesting a potential role in AML progression and the EMT process. CONCLUSIONS This study demonstrates that CDK9 is a potential prognostic biomarker and therapeutic target in AML. Its involvement in multiple key pathways during AML development and its influence on the tumor immune microenvironment support further exploration of CDK9-targeted therapies to improve AML treatment outcomes.