Posttranscriptional regulation plays a fundamental role in the biology of embryonic stem cells (ESCs). Many studies have demonstrated that multiple mRNAs are coregulated by one or more RNA-binding proteins (RBPs) that orchestrate mRNA expression. A family of RBPs, which is known as the Pumilio-FBF (PUF) family, is highly conserved among different species and has been associated with the undifferentiated and differentiated states of different cell lines. In humans, two homologs of the PUF family have been found: Pumilio 1 (PUM1) and Pumilio 2 (PUM2). To understand the role of these proteins in human ESCs (hESCs), we first assessed the influence of the silencing of PUM1 and PUM2 on pluripotency genes and found that the knockdown of Pumilio genes significantly decreased the OCT4 and NANOG mRNA levels and reduced the amount of nuclear OCT4, which suggests that Pumilio proteins play a role in the maintenance of pluripotency in hESCs. Furthermore, we observed that PUM1-and-PUM2-silenced hESCs exhibited improved efficiency of in vitro cardiomyogenic differentiation. Through an in silico analysis, we identified mRNA targets of PUM1 and PUM2 that are expressed at the early stages of cardiomyogenesis, and further investigation will determine whether these target mRNAs are active and involved in the progression of cardiomyogenesis. Our findings contribute to the understanding of the role of Pumilio proteins in hESC maintenance and differentiation.