Embryonic stem cells (ESCs) are pluripotent stem cells characterized by their ability to self-renew and their pluripotency to differentiate into all cell types. MicroRNA (miRNA) is a small non-coding RNA molecule which can regulate transcriptional and post-transcriptional gene expression, and may also play significant roles in regulating proliferation and differentiation of ESCs. The maintenance of pluripotency in ESCs may involve a regulatory network of many factors and pathways regulated by miRNA, which includes ESCs transcription factors, cell cycle regulation, epigenetic modifications as well as intracelluar signal transduction. This review mainly elaborates the biogenesis of miRNA, the miRNA families regulating the pluripotency of ESCs, and the effect of miRNA on the regulatory network of pluripotency in ESCs.