The transcription factor hepatocyte nuclear factor-1β (HNF-1β) regulates tissue-specific gene expression in the kidney and other epithelial organs. Mutations of HNF-1β produce kidney cysts, and previous studies have shown that HNF-1β regulates the transcription of cystic disease genes, including Pkd2 and Pkhd1. Here, we combined chromatin immunoprecipitation and next-generation sequencing (ChIP-Seq) with microarray analysis to identify microRNAs (miRNAs) that are directly regulated by HNF-1β in renal epithelial cells. These studies identified members of the epithelial-specific miR-200 family (miR-200b/200a/429) as novel transcriptional targets of HNF-1β. HNF-1β binds to two evolutionarily conserved sites located 28 kb upstream to miR-200b. Luciferase reporter assays showed that the HNF-1β binding sites were located within a promoter that was active in renal epithelial cells. Mutations of the HNF-1β binding sites abolished promoter activity. RT-PCR analysis revealed that a long noncoding RNA (lncRNA) is transcribed from the promoter and encodes the miR-200 cluster. Inhibition of the lncRNA with siRNAs decreased the levels of miR-200 but did not affect expression of the Ttll10 host gene. The expression of the lncRNA and miR-200 was decreased in kidneys from HNF-1β knock-out mice and renal epithelial cells expressing dominant-negative mutant HNF-1β. The expression of miR-200 targets, Zeb2 and Pkd1, was increased in HNF-1β knock-out kidneys and in cells expressing mutant HNF-1β. Overexpression of miR-200 decreased the expression of Zeb2 and Pkd1 in HNF-1β mutant cells. These studies reveal a novel pathway whereby HNF-1β directly contributes to the control of miRNAs that are involved in epithelial-mesenchymal transition and cystic kidney disease.