Chronic lymphocytic leukemia (CLL) is a highly heterogeneous disease. Despite recent tremen-dous progress in managing CLL, the disease remains incurable with clinical therapies, and relapse is inevitable. To overcome this, new diagnostic and prognostic markers need to be investigated. We thus screened through the public database for genes with diagnostic, prognostic, and therapeutic implications in CLL. We further performed RT-qPCR and Western blot analysis to measure the candidate gene and protein expression levels, respectively, in peripheral blood mononuclear cells. Our results indicated that Glyoxalase 1 (GLO1) expression was significantly higher in patients with CLL than in healthy controls. Furthermore, cell proliferation, apoptosis, and cell cycle assay results together indicated that S-p-bromobenzylglutathione cyclopentyl diester (BBGC), an effective inhibitor of GLO1, suppresses the progression of CLL. Bioinformatics analysis revealed that GLO1 expression is closely associated with CDK4 expression in a wide variety of cancer types, and inhibition of CDK4 through silencing of genes or inhibitors can downregulate GLO1 expression. Subsequent validation experiments demonstrated that GLO1 protein levels were downregulated in MEC-1 and Jurkat cell lines after palbociclib exposure, and combination treatment of palbociclib with GLO1 inhibitor BBGC effectively delayed the growth of tumor cell lines. Chronic lymphocytic leukemia is a highly heterogeneous disease. Despite recent tremen-dous progress in managing CLL, the disease remains incurable with clinical therapies, and relapse is inevitable. In this context, our results provide new insights about GLO1 and CDK4 expression dysregulation in CLL and its role promoting tumor progression. • Our data suggest that GLO1 protein inhibitor exhibited potent therapeutic potential in blocking cell survival and triggering cell cycle arrest in CLL cell lines. • We found that GLO1 protein expression was reduced after inhibiting CDK4, implying that the cell cycle arrest targeting the CDK4 signaling pathway may be related to GLO1 protein. • The results of the drug experiments suggest that the combination of BBGC and palbociclib deserves attention and further study. [ABSTRACT FROM AUTHOR]