Abstract: This study investigated the effects of prolonged subculture on Atractylodes lancea tissue culture seedlings by examining morphological, physiological, biochemical, and transcriptomic changes across the third, tenth, and seventeenth subculture generations. Results indicated that: (1) With increasing subculture generations, growth rates declined, and seedlings exhibited reduced biomass, height, and leaf length-to-width ratios, indicative of phenotypic degradation. (2) Telomerase reverse transcriptase activity and chlorophyll fluorescence parameters (F_o and F_m) decreased, while soluble sugar, hydrogen peroxide (H₂O₂), malondialdehyde (MDA), and the activities of superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) increased. (3) Levels of endogenous hormones, including indole-3-acetic acid (IAA), cytokinin (CTK), and gibberellin (GA₃), also increased significantly with successive subcultures, indicating a progressive degree of hormonal dysregulation and degradation. (4) Transcriptome analysis revealed inhibition of fatty acid degradation, down-regulation of genes associated with the mevalonate (MVA) pathway, and blockade of acetyl coenzyme A synthesis. Genes associated with the gibberellin and ethylene pathways were down-regulated, while those related to the abscisic acid and auxin pathways were up-regulated. These disruptions in hormone metabolism and associated physiological processes contributed to metabolic imbalances and molecular-level degradation. Overall, prolonged subculture led to morphological atrophy, metabolic disturbances, accumulation of stress substances, inhibition of primary metabolic processes such as fatty acid degradation and secondary metabolite biosynthesis, and dysregulation of genes related to hormone signal transduction pathways. These combined effects culminated in the progressive degradation of A. lancea tissue culture seedlings.