Hepatocyte nuclear factor 4α (HNF4α) is a tissue-specific transcription factor known to regulate a large number of genes in hepatocytes and pancreatic β cells. Although HNF4α is highly expressed in some sections of the kidney, little is known about its role in this organ and about HNF4α-regulated genes in the kidney cells. The abundance and activity of HNF4α are frequently reduced in renal cell carcinoma (RCC) indicating some tumor suppressing function of HNF4α in renal cells. To determine the potential role of HNF4α in RCC, we used Flp recombinase-mediated gene integration to generate human embryonic kidney cells (HEK293) that conditionally express wild-type or mutated HNF4α. Expression of wild-type HNF4α but not of the mutants led to reduction of proliferation and alterations of cell morphology. These effects were reversible and induced at physiological concentrations of HNF4α. Using gene expression profiling by microarrays, we determined genes regulated by HNF4α. Interestingly, many of the genes regulated by HNF4α have been shown to be deregulated in RCC microarray studies. These genes (ACY1, WT1, SELENBP1, COBL, EFHD1, AGXT2L1, ALDH5A1, THEM2, ABCB1, FLJ14146, CSPG2, TRIM9 and HEY1) are good candidates for genes whose activity is changed upon the decrease of HNF4α in RCC.