Objective · To globally study the influence of arsenite to the various biological pathways of Escherichia coli as a model organism. Methods · The protein-arsenite interactions was globally studied based on a proteome microarray constructed by 4 256 affinity-purified Escherichia coli proteins. The functions of interacting proteins and their network were then analyzed by bioinformatics. Results · 91 proteins that remarkably interact with arsenic were successfully identified. Bioinformatics analysis found that most of the proteins possess catalytic activity and are involved in various biosynthesis and cellular metabolism pathways. The interactions of arsenic with proteins encoded by malY, cfa and hypF genes were further validated by Western blotting, which proves the results of proteome microarray reliable. Conclusion · Arsenite interacts with a variety of enzymes of Escherichia coli and can greatly affect its biological metabolism.