Hepatocyte nuclear factor 1β (HNF1β) is an essential transcription factor in development and function of the kidney, the pancreas and the liver. Mutations or deletions in HNF1β cause a heterogenous phenotype including renal cysts and/or malformation, maturity-onset diabetes of the young, liver malfunction and hypomagnesemia. The spectrum of HNF1β–regulated genes and pathways is only partially deciphered. Here, we aim to identify the HNF1β target genes and pathways that explain the multifaceted role of HNF1β in the kidney. RNA-sequencing was performed in a mouse distal convoluted tubule (DCT) cell line transfected with siRNAs targeting HNF1β or non-targeting siRNAs. A total number of 989 differential expressed genes was identified. Subsequently, HNF1β binding sites in the promoter regions (defined as <5-kb from the transcription start site) of these genes were identified using ChIP-sequencing data from the same cell line. Only 9,3% (92 genes) of the differentially expressed genes contained a HNF1β-binding site within their promoter region. These genes included known targets of HNF1β including Pkhd1, Kif12 and Kcnj16 and unknown targets. GO-term pathway analysis demonstrated that regulation of cell polarity, cell-cell junction complexes and cytoskeleton organization were regulated by HNF1β. Indeed, transepithelial electrical resistance measurements were reduced upon expression of dominant negative (DN) HNF1β. Nine HNF1 β targets that are part of the apical cell polarity complex were selected for further analysis. To study the importance of HNF1β in regulating cell polarity, the integrity of this apical cell polarity complex will be analyzed in kidney cells expressing wild-type HNF1β and DN HNF1β using immunocytochemistry. Together these findings suggest a role for HNF1β in a transcriptional network regulating cell polarity in adult kidney cells by transcriptional control of the apical polarity complex in epithelial cells.