Serum- and 2i-cultured embryonic stem cells (ESCs) show different epigenetic landscapes and transcriptomic profiles. The difference in the function and expression of microRNAs ( miRNAs) between these two states remains unclear. Here, we showed that 2i- and serum-cultured ESCs exhibited distinctive miRNA expression profiles with >100 miRNAs differentially expressed, and the expression changes were largely due to transcriptional regulation. We further characterized the function of miRNAs differentially expressed under two conditions and found that ESCs exhibited higher degree of dependency on miRNAs for rapid proliferation; since Dgcr8(-/-) or Dicer1(-/-) but not wild-type ESCs showed slower growth rate and more accumulation in the G1 phase under 2i than serum condition. More interestingly, introduction of various self-renewal-silencing miRNAs in wild-type or Dgcr8(-/-) ESCs failed to silence the self-renewal in 2i medium, but regained the ability to silence the self-renewal upon the addition of serum. Our findings reveal significant differences in the expression and function of miRNAs between serum-and 2i-cultured ESCs. Copyright (C) 2017, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, and Genetics Society of China. Published by Elsevier Limited and Science Press. All rights reserved.