Embryonic stem cells (ESCs) and embryonal carcinoma cells (ECCs) possess the remarkable property of self-renewal and differentiation potency. They are model preparations for investigating the underlying mechanisms of "stemness". microRNAs are recently discovered small noncoding RNAs with a broad spectrum of functions, especially in control of development. Here, we show that miR-302b indirectly regulates expression of the pluripotent stem cell marker Oct4, and it directly regulates expression of Cyclin D2 protein, a developmental regulator during gastrulation. Using loss-of function and gain-of function approaches, we demonstrate that functional miR-302b is necessary to maintain stem cell self-renewal and inhibit neuronal differentiation of human ECCs. During retinoic acid-induced neuronal differentiation, Cyclin D2 protein but not mRNA expression is strongly increased, concurrent with the down-regulation of miR-302b and Oct4. Our results suggest that miR-302b plays an important role in maintaining the pluripotency of ECCs and probably ESCs, by post-transcriptional regulation of Cyclin D2 expression.