It has been reported that the dynamic interplay between O-GlcNAcylation and O-phosphorylation is responsible for altering the activity or localization of heat-shock proteins. The aim of this study was to determine whether dynamic interplay between O-GlcNAcylation and O-phosphorylation of HSP27 in hepatocellular cancer (HCC) cells affect its entry into the nucleus. We demonstrate that the entry of HSP27 into the nucleus correlated with its phosphorylation through transfecting HCC cells with plasmids coding for wild-type HSP27 (HSP27-WT), its non-phosphorylatable (HSP27-3A) and pseudophosphorylated (HSP27-3D) mutants, however, not all of the endogenous or exogenous nuclear HSP27 was modified by phosphorylation. We observed that HSP27 was modified with O-GlcNAc glycosylation in HCC cells and report that at conserved Ser residues of HSP27, alternative phosphorylation and O-GlcNAc modification can be predicted by the YinOYang 1.2 method. Furthermore, after P79350 or combined SB203580 and PUGNAc treatment, increased nuclear import of HSP27-WT and HSP27-3D implied that the entry of HSP27 into the nucleus was not only correlated with phosphorylation, but also with O-GlcNAc glycosylation. Collectively, O-GlcNAcylation of HSP27 in HCC cells may be a novel regulatory mode of HSP27 function, particularly for its entry into the nucleus. Crosstalk or interplay between glycosylation and phosphorylation of HSP27 could regulate its subcellular localization and biological functions in liver cancer.