Interleukin-21 (IL-21) has pleiotropic functions on the cells, which play roles in both innate and acquired immunity, such as T cells, B cells, natural killer (NK) cells and dendritic cells. In this study we identified a novel isoform of IL-21, IL-21iso in human and mouse. IL-21iso might be an alternative splicing variant form and the C-terminal region of predicted IL-21iso amino acid sequences were different from original IL-21 in both human and mouse. In spite of the differences in C-terminal amino acid sequences, both human IL-21 and IL-21iso showed comparable proliferative effect on anti-CD40 Ab-activated primary B cells, anti-CD3 Ab-activated primary T cells and human NK cell line, NK0, and upregulated IFN-γ production from NK0. Furthermore IL-21 and IL-21iso similarly activated STAT1 and STAT3. IL-21iso mRNA was expressed in activated T cells as well as IL-21 mRNA. However, cycloheximide treatment partially blocked the upregulation of IL-21iso mRNA in activated T cells while little affected the IL-21 mRNA expression suggesting that de novo protein synthesis is required for the full expression of IL-21iso transcript. We also show that the secretion efficiency of hIL-21iso is much lower than that of hIL-21. These results may suggest there are some different regulatory mechanisms to produce IL-21 or IL-21iso in transcriptional and secretory steps.