Epigenetic modifications are known to play critical roles in the expression of genes related to differentiation and dedifferentiation. Histone lysine demethylase KDM5B (PLU-1) catalyzes the demethylation of histone H3 on Lys 4 (H3K4), which results in the repression of gene expression. KDM5B is involved in regulation of luminal and basal cell specific gene expression in breast cancers. However, the mechanisms by which KDM5B is regulated in breast cancer, in particular in response to post-translational signals is not well-defined. Here, we demonstrate that KDM5B is phosphorylated at Ser1456 by the cyclin-dependent kinase 1 (CDK1). Phosphorylation of KDM5B at Ser1456 attenuated the occupancy of KDM5B on the promoters of pluripotency genes. Moreover, KDM5B inhibited the expression of pluripotency genes, SOX2 and NANOG, and decreased the stem cell population in triple-negative breast cancer cell lines (TNBC). We previously reported that the tumor suppressor HEXIM1 is a mediator of KDM5B recruitment to its target genes, and HEXIM1 is required for the inhibition of nuclear hormone receptor activity by KDM5B. Similarly, HEXIM1 is required for regulation of pluripotency genes by KDM5B.