The Krüppel-like factor (Klf) gene family encodes transcription factors that play an important role in the regulation of stem cell proliferation, cell differentiation and development in bilaterians. Although Klf genes have been shown to specify functionally various cell types in non-bilaterian animals, their role in early-diverging animal lineages has not been assessed. Thus, the ancestral activity of these transcription factors in animal development is not well understood. The ctenophore Mnemiopsis leidyi has emerged as an important non-bilaterian model system for understanding early animal evolution. Here, we characterize the expression and functional role of Klf genes during M. leidyi embryogenesis. Zygotic Klf gene function was assessed with both CRISPR/Cas9-mediated genome editing and splice-blocking morpholino oligonucleotide knockdown approaches. Abrogation of zygotic Klf expression during M. leidyi embryogenesis resulted in abnormal development of several organs, including the pharynx, tentacle bulbs and apical organ. Our data suggest an ancient role for Klf genes in regulating endodermal patterning, possibly through regulation of cell proliferation.