<jats:title>Abstract</jats:title><jats:sec><jats:title>Objective</jats:title><jats:p>PDZ‐binding kinase (PBK) has been reported as a poor prognostic factor and is a promising molecular target for anticancer therapeutics. Here, we aimed to investigate the effect of specific PBK inhibitor OTS514 on the survival of OSCC cells.</jats:p></jats:sec><jats:sec><jats:title>Methods</jats:title><jats:p>Four OSCC cell lines (HSC‐2, HSC‐3, SAS, and OSC‐19) were used to examine the effect of OTS514 on cell survival and apoptosis. DNA microarray analysis was conducted to investigate the effect of OTS514 on gene expression in OSCC cells. Gene set enrichment analysis was performed to identify molecular signatures related to the antiproliferative effect of OTS514.</jats:p></jats:sec><jats:sec><jats:title>Results</jats:title><jats:p>OTS514 decreased the cell survival of OSCC cells dose‐dependently, and administration of OTS514 readily suppressed the HSC‐2‐derived tumor growt<jats:italic>h</jats:italic> in immunodeficient mice. Treatment with OTS514 significantly increased the number of apoptotic cells and caspase‐3/7 activity. Importantly, OTS514 suppressed the expression of E2F target genes with a marked decrease in protein levels of E2F1, a transcriptional factor. Moreover, <jats:italic>TP53</jats:italic> knockdown attenuated OTS514‐induced apoptosis.</jats:p></jats:sec><jats:sec><jats:title>Conclusion</jats:title><jats:p>OTS514 suppressed the proliferation of OSCC cells by downregulating the expression of E2F target genes and induced apoptosis by mediating the p53 signaling pathway. These results highlight the clinical application of PBK inhibitors in the development of molecular‐targeted therapeutics against OSCC.</jats:p></jats:sec>