Objective? To investigate whether Jianpi Huazhuo Formula (JPHF) improves hepatic lipid accumulation in non-alcoholic fatty liver disease (NAFLD) through the regulation of mitophagy and to elucidate its related molecular mechanisms.Method To establish a cellular steatosis model, HepG2 cells were exposed to sodium palmitate–sodium oleate (800/1 600 μmol·L?1, respectively) for 24 h. The cells were then treated with blank control medium, model medium, or medium supplemented with low-, medium- or high-dose JPHF-containing serum, or atorvastatin. Cell viability was assessed with the CCK-8 assay. Lipid accumulation was evaluated by Oil Red O and Nile Red staining and by measuring intracellular triglyceride (TG) levels. Differentially expressed genes and enriched pathways were identified by high-throughput sequencing followed by bioinformatic analysis. Protein levels of PINK1, Parkin, LC3-II/Ⅰ and p62 were determined by Western blotting. Mitochondrial membrane potential was monitored with the JC-1 fluorescent probe; autophagosome formation was visualized with adenovirus-mediated mCherry-GFP-LC3B. Ultrastructural changes were observed under transmission electron microscopy.Results? Compared with the model group, medium- and high-dose JPHF-containing serum significantly increased cell viability (P< 0.01) and decreased intracellular TG levels (P< 0.01,P< 0.001) and lipid droplet accumulation. High-throughput sequencing identified 1 402 differentially expressed genes; KEGG analysis revealed significant enrichment of the “mitophagy” pathway, with marked up-regulation of the key initiator PINK1. Mechanistic experiments demonstrated that JPHF dose-dependently elevated PINK1 and Parkin protein levels, raised the LC3-II/I ratio (P< 0.05，P< 0.01，P< 0.001), decreased p62 expression (P< 0.05，P< 0.01，P< 0.001), reduced abnormally high mitochondrial membrane potential (P< 0.05，P< 0.01), and increased the number of autophagosomes (P< 0.0001). Transmission electron microscopy further revealed typical double-membrane mitochondrial autophagosomes in the high-dose JPHF group.Conclusion? JPHF effectively alleviates hepatic steatosis by activating the PINK1/Parkin pathway and inducing mitophagy.