Using proteomics, proteins regulated by 17beta-estradiol (E2) were identified in human MCF-7 breast cancer cells; 26 proteins including heat shock proteins (Hsps) were differentially regulated by E2 in cells cultured in serum-free condition. When the transcript levels of these proteins and another Hsps were measured by real-time PCR, the transcripts encoding 6 proteins (Hsp56, Hsp90alpha, Hsp110, protein disulfide isomerase related protein, XTP3-transactivated protein A and stathmin 1) were significantly up-regulated and that encoding aminoacylase 1 was down-regulated by E2 in cells cultured with or without serum. The protein profiling and transcript expression patterns of E2-regulated proteins including Hsps in MCF-7 cells suggested the involvement of these proteins in breast carcinogenesis. Proteins (or transcripts) whose expression pattern is altered by E2 might be potent targets for treating breast cancer patients.