Oral squamous cell carcinoma (OSCC), with aggressive locoregional invasion, has a high rate of early recurrences and poor prognosis. Dihydroartemisinin (DHA), as a derivative of artemisinin, has been found to exert potent antitumor activity. Recent studies reported that DHA suppresses OSCC cell growth and viability through the regulation of reactive oxygen species (ROS) production and mitochondrial calcium uniporter. However, the mechanism underlying the action of DHA on OSCCs remains elusive. In the study, we observed that 159 genes were remarkably misregulated in primary OSCC tumors associated with DHA-inhibited pathways, supporting that OSCCs are susceptible to DHA treatment. Herein, our study showed that DHA exhibited promising effects to suppress OSCC cell growth and survival, and single-cell colony formation. Interestingly, the combination of DHA and cisplatin (CDDP) significantly reduced the toxicity of CDDP treatment alone on human normal oral cells (NOK). Moreover, DHA remarkably impaired mitochondrial structure and function, and triggered DNA damage and ROS generation, and activation of mitophagy. In addition, DHA induced leakage of cytochrome C and apoptosis-inducing factor (AIF) from mitochondria, elevated Bax/cleaved-caspase 3 expression levels and compromised Bcl2 protein expression. In the OSCC tumor-xenograft mice model, DHA remarkably suppressed tumor growth and induced apoptosis of OSCCsin vivo. Intriguingly, a selective mitophagy inhibitor Mdivi-1 could significantly reinforce the anticancer activity of DHA treatment. DHA and Mdivi-1 can synergistically suppress OSCC cell proliferation and survival. These data uncover a previously unappreciated contribution of the mitochondria-associated pathway to the antitumor activity of DHA on OSCCs. Our study shed light on a new aspect of a DHA-based therapeutic strategy to combat OSCC tumors.