Cervical cancer is one of the leading causes of cancer death in women (Munoz et al., 2004), and current treatment strategies though effective still possess limitations. Thus, identification of more molecular-targeted therapeutics for cervical cancer is urgently needed. We recently identified an isoform of mixed lineage leukemia 5 (MLL5β, 503 amino acids) that is specifically expressed in HPV16/18+ cervical cancer cell lines and patient samples (Yew et al., 2011). MLL5β was found to regulate E6/E7 oncogene transcription activation through its interaction with AP-1 at the 5′ segment of the HPV16/18-long control region (LCR). With no known DNA-binding motifs on MLL5β, interaction of MLL5β with AP-1 (c-Fos/c-Jun, AP-1 dimer) was thought to be essential for its recruitment to the HPV16/18-LCR (Yew et al., 2011). As E6 and E7 are the main pathological factors of HPV16/18+ cervical cancer, the development of new therapeutics for this type of cancer has been focused on the inhibition of E6/E7 expression (Butz et al., 2003; Putral et al., 2005). The identification of the essential role of MLL5β in E6/E7 transcriptional regulation (Yew et al., 2011) poses MLL5β as an attractive molecular target. A further understanding of the molecular mechanisms underlying recruitment and assembly of the MLL5β–AP-1 transcriptional activation complex to the HPV16/18-LCR could provide new insights into MLL5β-targeted therapeutics. Here, we report the essential role of MLL5β O-GlcNAcylation in MLL5β–AP-1 complex assembly at the HPV16/18-LCR and the selective cytotoxic effects of O-GlcNAcylation inhibition on HPV16/18+ cervical cancer cells. Our findings highlight the potential of targeting MLL5β O-GlcNAcylation as a novel therapeutic approach for HPV16/18+ cervical cancer.