MicroRNAs (miRNAs) are endogenous small non-coding RNAs which decrease the expression levels of specific genes by translational repression, sequestration and degradation of their mRNAs. Angiotensin II is an important modulator of adrenal zona glomerulosa cell physiology, including steroidogenesis and proliferation among many other physiological processes. Since each miRNA may regulate the expression levels of multiple genes, thereby resembling the transcription regulatory networks triggered by transcription factors, we hypothesize that specific miRNAs may be involved in angiotensin II-mediated adrenocortical cell physiology. The human adrenocortical cell line H295R is the only adrenal cell line available with a steroid secretion pattern and regulation similar to freshly isolated adrenocortical cells. We screened for miRNAs regulated by angiotensin II in H295R cells and found that mir-21 expression levels were specifically modulated by angiotensin II. Angiotensin II-time dependently increased mir-21 expression reaching a 4.4-fold induction after 24 h. Angiotensin II-mediated mir-21 expression resulted in biologically active mir-21, determined using a fusion mRNA reporter system carrying mir-21 target sequences in its 3' UTR. Upregulation of mir-21 intracellular levels increased aldosterone secretion but not cortisol. Elevation of mir-21 levels also increased cell proliferation in H295R cells. In summary, mir-21 is an endogenously expressed miRNA in human adrenal cells. Mir-21 expression is upregulated by angiotensin II and its overexpression caused an increase in aldosterone secretion and cell proliferation. Alterations in mir-21 expression levels or function may be involved in dysregulation of angiotensin II signaling and abnormal aldosterone secretion by adrenal glands in humans.