A capability to proliferate under the three-dimensional (3D) conditions is a characteristic property of cancer. Therefore, factors that regulate the 3D growth are considered rational targets for cancer therapy. We analyzed gene expression profile of human prostate cancer PC3 cells which were cultured in normal 2D conditions or in spheroid culture conditions (3D). The analysis suggested that the sheroid conditions would mimic a part of tumor-like 3D conditions. In addition, we analyzed genes regulated by TRIB1, a candidate oncogene in PC3 cells by comparing the gene expression profiles between control (shGFP)-treated and TRIB1 shRNA- (shTRIB1-1 and shTRIB1-2) treated cells. Our analysis identified a subset of genes that are downstream of TRIB1 in prostate cancer cells.