Our previous studies have shown that genistein inhibits the growth of PC3 prostate cancer cells and induces apoptosis by inhibiting nuclear factor kappaB (NF-kappaB) and Akt signaling pathways. To better understand the precise molecular mechanism(s) by which genistein exerts its effects on PC3 cells, we utilized cDNA microarray to interrogate 12,558 known genes to determine the gene expression profiles altered by genistein treatment. We found a total of 832 genes that showed a greater than twofold change after genistein treatment from two independent experiments with a high degree of concordance. Among these genes, 774 genes were down-regulated and 58 genes were up-regulated with genistein treatment. Cluster analysis showed nine different types of expression alternations. These genes were also subjected to cluster analysis according to their biological functions. We found that genistein regulated the expression of genes that are critically involved in the regulation of cell growth, cell cycle, apoptosis, cell signaling transduction, angiogenesis, tumor cell invasion and metastasis. Reverse transcription-polymerase chain reaction (RT-PCR) analysis was used to confirm the results of cDNA microarray, and the results of RT-PCR were consistent with the microarray data. We conclude that genistein affected the expression of a large number of genes that are related to the control of cell survival and physiologic behaviors. The gene expression profiles provide comprehensive molecular mechanism(s) by which genistein exerts its pleiotropic effects on cancer cells. Genistein-induced regulation of these genes may be further exploited for devising chemopreventive and/or therapeutic strategies for prostate cancer.