Background The existence of tumor initiating cells (TICs) has been described in head and neck cancers (HNCs). Plasminogen activator inhibitor-1 (PAI-1) has been demonstrated to act as a prognostic factor in HNCs. Methods Tiplaxtinin (PAI-039), a specific inhibitor of PAI-1, and PAI-1 specific siRNA were used to examine the role of PAI-1 in the self-renewal property of HNC-TICs by tumorsphere formation. Western blot, real-time PCR, and luciferase-based reporter assay were used to study the effect of PAI-039 in the Sox2 expression. Results PAI-039 suppressed the self-renewal capability of HNC-TICs derived from HNC cell lines through the inhibition of Sox2 expression. PAI-039 decreased the activity of the core promoter and the enhancer of Sox2 gene in HNC-TICs. Knockdown of PAI-1 expression also inhibited self-renewal and radioresistance properties of HNC-TICs. Conclusion The inhibition of PAI-1 by PAI-039 or siRNA could suppress HNC-TICs within HNC cell lines through the down-regulation of Sox2. This article is protected by copyright. All rights reserved.