Migraine is the second highest cause of disability worldwide, bringing a huge socioeconomic burden. Improving mitochondrial function has promise as an effective treatment strategy for migraine. Szeto-Schiller peptide (SS-31) is a new mitochondria-targeted tetrapeptide molecule that has been shown to suppress the progression of diseases by restoring mitochondrial function, including renal disease, cardiac disease, and neurodegenerative disease. However, whether SS-31 has a therapeutic effect on migraine remains unclear. The aim of this study is to clarify the treatment of SS-31 for headache and its potential mechanisms. Here we used a mouse model induced by repeated dural infusion of inflammatory soup (IS), and examined roles of Sirt3/Pgc-1α positive feedback loop in headache pathogenesis and mitochondrial function. Our results showed that repeated IS infusion impaired mitochondrial function, mitochondrial ultrastructure and mitochondrial homeostasis in the trigeminal nucleus caudalis (TNC). These IS-induced damages in TNC were reversed by SS-31. In addition, IS-induced nociceptive responses were simultaneously alleviated. The effects of SS-31 on mitochondrial function and mitochondrial homeostasis (mainly mitochondrial biogenesis) were attenuated partially by the inhibitor of Sirt3/Pgc-1α. Overexpression of Sirt3/Pgc-1α increased the protein level of each other. These results indicated that SS-31 alleviated nociceptive responses and restored mitochondrial function in an IS-induced headache mouse model via Sirt3/Pgc-1α positive feedback loop. SS-31 has the potential to be an effective drug candidate for headache treatment.