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miR-26b has been implicated in a wide range of human diseases, including cancer, diabetes, heart disease, Alzheimer’s disease and osteoarthritis. To provide a tool to explore the importance of miR-26b in this broad context, we have generated and characterized a homozygous miR-26b stem-loop knockout human iPSC line. This gene-edited line exhibited a normal karyotype, expressed pluripotency markers and differentiated into cells representative of the three embryonic germ layers. This iPSC line will be valuable for studies investigating disease mechanisms and testing therapeutic strategies in vitro.