Aphids exclusively feed on plant phloem sap that contains much sugar and some non-essential amino acids but is poor in lipids and proteins. Conventionally, it has been believed that aphids substantially have no intestinal digestion of proteins. However, we here report an unexpected finding that cysteine protease genes of the family cathepsin B are massively amplified in the lineage of aphids, and that many of the protease genes exhibit gut-specific over-expression. By making use of EST data, sequenced cDNAs, and genomic trace sequences of the pea aphid Acyrthosiphon pisum, we identified a total of 28 cathepsin B-like gene copies in the genome of A. pisum. Phylogenetic analyses of all the cathepsin B genes in aphids revealed that genic expansion has continuously proceeded with basal, intermediary and recent duplications. Estimation of molecular evolutionary rates indicated that major alterations of the rates often occurred after duplications. For example, a gene copy ("348") was shown to be slow-evolving and close to genes of other insects like Drosophila melanogaster, while the other gene copies appeared to have evolved faster with higher ratios of non-synonymous to synonymous substitutions. We identified a number of gene copies (16 in A. pisum) that contained a replacement at the site required for catalytic activity of the protease. Among these, two copies were pseudogenes, while the remaining copies were structurally intact and possibly acquired new functions. For example, a cluster of such gene copies ("1674") has been subjected to positive selection. Quantitative RT-PCR analyses revealed that the more conserved gene copy ("348") showed a constitutive expression while five other forms ("84", "16", "16D", "1874" and "2744") were preferentially expressed in the gut of A. pisum. Putative biological roles of the diversified cathepsin B-like gene copies in aphids are discussed in relation to their nutritional physiology specialized for plant sap feeding lifestyle.