--- - |2- Abstract Interleukin‐21 (IL‐21) is a cytokine with potent regulatory effects on different immune cells. Recently, IL‐21 has been contemplated for use in the treatment of cancers. However, the molecular mechanisms regulating human IL‐21 gene expression has not yet been described. In this study, we initially studied the promoter region and identified the transcription start site. We thereafter described the essential region upstream of the transcription start site and showed the in vivo binding of NFATc2 and SP1 transcription factors to this region, in addition to their positive role in IL‐21 expression. We also studied the role of microRNAs (miRNAs) in regulating IL‐21 expression. We, thus, established the miRNA profile of CD4+CD45RO+ versus CD4+CD45RA+ isolated from healthy volunteers and identified a signature composed of 12 differentially expressed miRNAs. We showed that miR‐302c is able to negatively regulate IL‐21 expression by binding directly to its target site in the 3′‐untranslated region. Moreover, after using fresh human CD4‐positive T cells, we observed the high acetylation level of histone H4, an observation well in line with the already described high expression of IL‐21 in CD4+CD45RO+ versus CD4+CD45RA+ T cells. Altogether, our data identified different molecular mechanisms regulating IL‐21 expression. - Journal of Cellular Physiology, EarlyView.