The differential expression of genes and related proteins of multidrug resistance in chemoresistant prostate cancer cell lines were elucidated in this study. RNA extracted from doxorubicin-resistant rat prostate cancer (PCa) cells (AT3/ADR1000) and native PCa cells was hybridized to expression arrays containing cDNAs from 588 known genes. Differential expression of selected genes was confirmed by quantitative reverse-transcription polymerase chain reaction (RT-PCR) analysis. Protein contents were measured by fluorescent flow cytometry and immunoblotting. Localization of selected proteins in cells was observed by immunocytochemical staining. Up-regulation of eleven genes and down-regulation of one single gene were displayed in the chemoresistant prostate cancer cells. Overexpression of mRNAs in macrophage migration inhibitory factor (MIF), DNA binding protein inhibitor 1 (ID1), and glutathione S-transferase-pi (GST-pi) were confirmed by gene-specific RT-PCR. Protein over-expression of GST-pi, MIF, and ID1 in resistant cells were 3.3-, 1.5-, and 1.5-fold to native cells, respectively. Immunocytochemistry revealed that GST-pi, MIF, and ID1 were present primarily in the cytoplasm of tumor cells, but ID1 also could be found in the nucleus. AT3/ADR1000 drug-resistant PCa cells displayed significantly increased expression of GST-pi, MIF, and ID1 proteins when compared with native PCa cells. It indicates these genes may play a role in drug resistance of prostate cancer.