The gut-enriched Krüppel-like factor (GKLF) is a recently identified eukaryotic transcription factor that contains three C₂H₂ zinc fingers. The amino acid sequence of the zinc finger portion of GKLF is closely related to several Krüppel proteins, including the lung Krüppel-like factor (LKLF), the erythroid Krüppel-like factor (EKLF) and the basic transcription element binding protein 2 (BTEB2). The DNA sequence to which GKLF binds has not been definitively established. In the present study we determined the DNA binding sequence of GKLF using highly purified recombinant GKLF in a target detection assay of an oligonucleotide library consisting of random sequences. Upon repeated rounds of selection and subsequent characterization of the selected sequences by base-specific mutagenesis a DNA with the sequence 5′-^G/_A^G/_AGG^C/_TG^C/_T-3′ was found to contain the minimal essential binding site for GKLF. This sequence is present in the promoters of two previously characterized genes: the CACCC element of the β-globin gene, which interacts with EKLF, and the basic transcription element (BTE) of the CYP1A1 gene, which interacts with Sp1 and several Sp1-like transcription factors. Moreover, the selected GKLF binding sequence was capable of mediating transactivation of a linked reporter gene by GKLF in co-transfection experiments. Our results establish GKLF as a sequence-specific transcription factor likely involved in regulation of expression of endogenous genes.