Cognitive dysfunction after propofol anesthesia has been previously found. The underlying mechanisms of this sequel remain unclear. Insoluble proteins as major targets of anesthetics participated in various pathophysiological processes. This study aimed to provide evidence that changes in insoluble proteome in rat hippocampus may be involved in molecular mechanism of cognitive dysfunction following propofol anesthesia. Proteins extracted from rat hippocampus were separated by two-dimensional electrophoresis (2-DE). Their expression patterns were observed at 1, 6, 24 h and 7 days after 3 h of propofol anesthesia. Differentially expressed protein spots among groups were submitted to matrix-assisted laser desorption/ionization time of flight mass spectrometer (MALDI-TOF MS) assay and peptide mass fingerprinting (PMF) identification. Identified proteins were further analyzed through Gene Ontology (GO). Results of 2-DE were selectively assayed using Western blot and RT-PCR. Fifty-nine differentially expressed proteins were detected, among which 43 were identified through MALDI-TOF MS. Most identified proteins were distributed in organelles and membranes. According to biological process category, 27 proteins were involved in metabolic process, 19 in developmental process, 14 in stimulus-response, and 21 in biological regulation. Most changes took place within 24 h, with more down-regulation within 6 h. Twelve proteins did not restore to the basic level until the 7th day after propofol anesthesia. Expressions of insoluble proteome dynamically changed following propofol anesthesia. Down-regulations at early stage might produce depressive effects, which may be involved in molecular mechanism of cognitive dysfunction after propofol anesthesia.