Abstract: OBJECTIVE This study aims to dynamically analyze the molecular adaptation mechanisms of biofilm development of Acinetobacter baumannii, providing theoretical basis and potential targets for the development of stage-specific anti-biofilm strategies. METHODS Through mass spectrometry-based quantitative proteomics technology, the global protein expression profiles of A. baumannii biofilm at different stages of development were compared systematically. RESULTS Temporal proteomic analysis revealed that during the initial colonization stage (0-24 h), bacteria downregulated ribosome synthesis and aerobic metabolism, while activating signal transduction systems to adapt to attachment. In the rapid growth stage (24-48 h), bacteria initiated anaerobic respiration and improved virulence factor phospholipase C, pyocyanin iron acquisition and DNA repair capabilities, while suppressing amino acid synthesis to enter a persistent state. By the maturation stage (48-72 h), the ureotelic metabolism pathway was significantly activated to cope with microenvironmental acidification and nitrogen source depletion, while growth activities like cell wall synthesis were inhibited, entering a stable maintenance phase. During the detachment phase (72-96 h), bacteria significantly downregulated ribosomes and energy-consuming secretion by improving acid phosphatase and antioxidant capacity, forming a highly tolerant phenotype. Meanwhile, throughout the entire biofilm development process, ubiquinone/terpenoid synthesis and DNA repair pathways were maintained, while exogenous aromatic degradation was weakened to concentrate resources for ensuring energy and genetic stability. CONCLUSIONS Temporal metabolic reprogramming serves as an intrinsic driving force for A. baumannii biofilm to achieve structural stability and high tolerance. The stage-specific key pathways and proteins revealed in this study provide new targets for precise intervention targeting specific developmental stages of biofilm.