Autopolyploidy and allopolyploidy are common in many plants and some animals. Rapid changes in genomic composition and gene expression have been observed in both auto- and allopolyploids, but the effects of polyploidy on proteomic divergence are poorly understood. Here we report quantitative analysis of protein changes in leaves of Arabidopsis auto- and allotetraploids and their progenitors using isobaric tags for relative and absolute quantitation (iTRAQ) coupled with mass spectrometry. Over 1 000 proteins analyzed, the levels of protein divergence were relatively high (~18%) between Arabidopsis thaliana and Arabidopsis arenosa, relatively low (~6.8%) between an A. thaliana diploid and autotetraploid, and intermediate (~8.3% and 8.2%) in F1 and F8 resynthesized allotetraploids relative to the mid-parent values, respectively. This pattern of proteomic divergence was consistent with the previously reported gene expression data. In particular, many nonadditively accumulated proteins (61-62%) in the F1 and F8 allotetraploids were also differentially expressed between the parents. The differentially accumulated proteins in functional categories of abiotic and biotic stresses were overrepresented between an A. thaliana autotetraploid and diploid and between two Arabidopsis species, but not significantly different between the allotetraploids and their progenitors. Although the trend of changes is similar, the percentage of differentially accumulated proteins that matched previously reported differentially expressed genes was relatively low. Western blot analysis confirmed several selected proteins with isoforms whose cumulative levels were differentially expressed. These data suggest high protein divergence between species as well as rapid changes in post-transcriptional regulation and translational modifications of proteins during polyploidization.