We demonstrate the use of an antibody (Ab) microarray for a comparative expression profiling of proteins in an L-threonine biosynthetic pathway of Escherichia coli between a parental strain (W3110) and L-threonine overproducing mutant (TF5015). On the basis of a global comparative transcriptome analysis between the two strains, 28 analytical target proteins were selected and subjected to a production of polyclonal Abs against them. An Ab microarray was constructed by spotting a set of produced antibodies on a glass slide, and was employed for a comparative expression profiling of the proteins between the two strains by a two-color fluorescence assay method. The performance of the Ab microarray was evaluated with respect to cross-reactivity of the antibodies, dye-labeling efficiency, and the nature of antigenic proteins. Of these, the cross-reactivity of the used antibodies was found to mainly cause the deviation of the observed expression ratios from the expected ones. To offset the deviations, correction factors were derived from a statistical analysis and introduced. As a result, ten proteins were categorized to be up-regulated, while one was down-regulated in TF5015. Expression profiling of proteins using the Ab microarray was further verified by comparison with Western blotting and 2-DE.