Oral squamous cell carcinoma (OSCC) is the most common malignancy in the oral cavity, which accounts for >90% of all diagnosed oral cancers. 2-phenylethynesulfonamide (PES) was known as a selective heat shock protein 70 (Hsp70) function inhibitor, which induced cytotoxic effects on various tumor cell types, but showed to be less toxic to normal cells. However, no associated evaluation of PES on OSCC was found. In the present study, the proliferation of OSCC cells treated with PES was analyzed using a CCK-8 assay. The effects of PES on the cell cycle and apoptosis of OSCC cells were determined by flow cytometric analyses. Expression of associated protein was determined by Western blot analysis. The results of the present study showed that PES inhibited the proliferation of OSCC cell lines in vivo and in vitro. PES induced apoptosis and arrested the cell cycle of OSCC cells. PES inhibited the expression of X-linked inhibitor of apoptosis protein (XIAP), baculoviral IAP repeat containing 2 (c-IAP1), phosphorylated AKT (p-AKT), and phosphorylated extracellular signal-regulated kinase (p-ERK). Additionally, knockdown of Hsp70 enhanced the effects of PES. By contrast, overexpression of Hsp70 attenuated the inhibitory effects of PES on cell viability. PES disrupted the interaction between Hsp70 and XIAP. In conclusion, the present study demonstrated that PES suppresses the growth of OSCC cells through Hsp70-dependent mechanism.