Mutations in the HNF1B gene cause autosomal dominant tubulointerstitial kidney disease (ADTKD)-HNF1β, a rare and heterogenous disease characterized by renal cysts and/or malformation, maturity-onset diabetes of the young, hypomagnesemia and hypokalemia. The electrolyte disturbances may develop in the distal part of the nephron, which is important for finetuning of Mg2+ and Ca2+ reabsorption. Therefore, we aim to study the transcriptional network directed by HNF1β in the distal part of the nephron. We combined HNF1β ChIP-seq and mRNA expression data to identify direct targets of HNF1β in a renal distal convoluted tubule cell line (mpkDCT). GO-term pathway analysis demonstrated enrichment of cell polarity, cell-cell junction and cytoskeleton pathways in the dataset. Genes directly and indirectly regulated by HNF1β within these pathways included members of the apical and basolateral polarity complexes including Crb3, Pard6b and Llgl2. Indeed, tight junction integrity was impaired in confluent mouse inner medullary collecting duct 3 (mIMCD3) cell monolayers expressing dominant negative Hnf1b compared to wildtype cells as observed by reduced transepithelial electrical resistance values and increased permeability for fluorescein (0.4 kDa). Expression of dominant negative Hnf1b also led to a decrease in height (30%), and increase in surface (58.5%) of cells grown on membranes. Moreover, 3D spheroids formed by cells expressing dominant negative Hnf1b were reduced in size compared to wildtype spheroids (30%). Together, these findings demonstrate that HNF1β directs a transcriptional network regulating tight junction integrity and cell structure in the distal part of the nephron.