Bacteria living in marine sediment play crucial roles in the benthic-pelagic interface coupling process. However, the complexity of the marine environment and the abundance of interfering materials hamper metaproteomic research of the marine mudflat environment. In this study, a modified sequential protein extraction method was used for marine mudflat sediment metaproteomic investigation. For marine sediment samples in cultured clam mudflat, more than 1000 protein spots were visualized in a two-dimensional gel electrophoresis map and 78 % of 194 randomly selected spots were successfully identified by mass spectrometry. We further applied this method to compare long-term clam aquaculture and natural mudflat sediment and identified 53 altered proteins from different microbe resources, which belonged to different functional categories or metabolic pathways. We found that proteins involved in stress/defense response process, ATP regeneration and protein folding more inclined to increase abundance while arginine biosynthesis and signal transduction process related proteins preferred to decrease in clam cultured mudflat sediment. Meanwhile, proteins were abundant in pathogens of bivalves, such as Vibrio and Photobacterium, and decreased in Acinetobacter, after about 8 months clam cultured. Furthermore, the terminal restriction fragment length polymorphism assay was performed to compare microbial community composition between sediments mentioned above. Results showed that the top three enrich genera in natural sediment were Cytophaga, Butyrivibrio and Spirochaeta, while Cytophaga, Spirochaeta and Azoarcus were found enrichment in long-term mudflat aquaculture sediment.