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Abstract: Periodontitis is a common oral disease caused by bacteria coupled with an excessive host immune response. Stem cell therapy can be a promising treatment strategy for periodontitis, but the relevant mechanism is complicated. This study aimed to explore the therapeutic potential of mitochondria from human embryonic stem cell-derived mesenchymal stem cells (hESC-MSCs) for the treatment of periodontitis. The gingival tissues of periodontitis patients are characterized by abnormal mitochondrial structure. Human gingival fibroblasts (HGFs) were exposed to 5 μg/mL of lipopolysaccharide (LPS) for 24 h to establish a cell injury model. Either when treated with hESC-MSCs or with mitochondria derived from hESC-MSCs, HGF showed a reduced expression of inflammatory genes, increased ATP levels, decreased ROS production, and enhanced mitochondrial function compared to the control. The average efficiency of isolated mitochondrial transfer by hESC-MSCs was determined to be 8.93%. Besides, a therapy of local mitochondrial injection in mice with LPS-induced periodontitis showed a reduction in inflammatory gene expression, as well as an increase in both mitochondrial number and aspect ratio in gingival tissues. In conclusion, our results indicate that mitochondria derived from hESC-MSCs can reduce the inflammatory response and improve mitochondrial function in HGFs, showcasing that the transfer of mitochondria between hESC-MSCs and HGFs serves as a potential mechanism underlying the stem cell therapeutic effect.