Background: Cytotoxic and pro-apoptotic effects exerted by the histone deacetylase inhibitor ITF2357 have been reported in acute myeloid leukemia HL-60 cells. In the current study, its mechanism of action was investigated at the molecular level. Materials and Methods: Cell proliferation was evaluated by methyl thiazol tetrazolium bromide reduction; apoptosis by annexin V, mitochondrial transmembrane potential by tetramethylrhodamine ethyl ester. Functional experiments and gene expression evaluations were performed by flow cytometry, microarray, and quantitative polymerase chain reaction. Results: Significant cell growth inhibition and increased apoptosis were observed. ITF2357 reduced protein levels of BCL-2, MCL-I, and BCL-X, and increased levels of BAK. Exposure to ITF2357 did not abrogate NF-kappa B DNA binding. After microarray analysis, interleukin-10, interleukin-6, epidermal growth factor, peroxisome prohferator-activated receptor (PPAR), transforming growth factor beta, P38 mitogen-activated protein kinase, aryl hydrocarbon receptor, xenobiotic metabolism, PPAR/retinoic acid receptor, NF-kappa B, apoptosis, lipopolysaccharide/interleukin-1, G-protein receptor, T-cell receptor, and platelet-derived growth factor were the de-regulated pathways. Conclusion: This study shows that ITF2357 influences both proliferation and inflammatory pathways in HL-60 cells; this observation could have possible applications in clinical practice.