AbstractmiR-204 has been proposed to modulate insulin expression in human pancreatic islets by regulating the expression of the MAFA transcript, and in turn insulin transcription. We investigated miR-204 expression in pancreatic endocrine tumors (PET), a panel of human tissues, tissues derived from pancreatic islet purification, and in induced pluripotent stem cells (iPSCs) differentiated towards a pancreatic endocrine phenotype by quantitative real time RT-PCR or droplet digital PCR (ddPCR). In addition, we evaluated the effect of miR-204 up- or down-regulation in purified human islets and in the EndoC-βH1 cell line, as an experimental model of human pancreatic β cells. Our results confirm that miR-204 was enriched in insulin producing PET, in β cells within healthy pancreatic islets, and highly expressed in EndoC-βH1 cells. Moreover, in iPSCs miR-204 increased stepwise upon stimulated differentiation to insulin producing cells. However, up- or down-regulation of miR-204 in human islets and in EndoC-βH1 cells resulted in modest and not significant changes of the MAFA and INS mRNAs measured by ddPCR or c-peptide release. Our data confirm the association of miR-204 with a β cell endocrine phenotype in human pancreatic islets, but do not support its direct role in regulating the levels of insulin mRNA through MAFA.