Objectives: The NLRP3 inflammasome-mediated pyroptosis in gingival fibroblasts (GFs) has been implicated in the pathogenesis of periodontitis. Mitochondrial dysfunction represents a critical upstream event in NLRP3 inflammasome activation, while mitophagy functions as an essential cellular mechanism for maintaining mitochondrial homeostasis. Therefore, the present study was designed to elucidate the regulatory role and molecular mechanisms of mitophagy in modulating pyroptosis in GFs. Materials and methods: Human GFs were used in this study. An in vitro inflammatory environment was established using 5 μg/ml lipopolysaccharide (LPS). GFs exposed to LPS were treated with 10 μM P62-mediated mitophagy inducer (PMI), while healthy GFs not exposed to LPS received 10 μM Mdivi-1 intervention. Mitophagy was visualized by immunofluorescence, and the expression levels of PINK1, Parkin, and Beclin-1 were assessed via qRT-PCR and Western blot. Mitochondrial morphology was examined by Transmission electron microscopy (TEM), and mitochondrial damage was evaluated through measurements of mitochondrial membrane potential (MMP) and intracellular reactive oxygen species (ROS) and mitochondrial ROS (mtROS) levels using flow cytometry. NLRP3 inflammasome activation was analyzed by qRT-PCR and Western blot. Propidium iodide (PI) staining, lactate dehydrogenase (LDH) release, IL-1β and IL-18 expression levels and apoptosis assays were also detected to assess pyroptosis in GFs. Results: LPS stimulation suppressed mitophagy in GFs, which was reversed by mitophagy activator. In contrast, mitophagy inhibition decreased basal mitophagy in healthy GFs. Mitophagy activation maintained mitochondrial function by enhancing MMP and reducing ROS/mtROS accumulation and mtDNA/nDNA ratio in LPS-stimulated GFs. Furthermore, mitophagy attenuated LPS-induced NLRP3 inflammasome activation and decreased GF death, and IL-1β/IL-18 production and secretion. Conclusions: Mitophagy alleviates NLRP3 inflammasome-dependent pyroptosis of GFs by maintaining mitochondrial homeostasis.