Ptf1a, also known as p48, is an important bHLH transcriptional factor, functioning as an integral part of a trimeric protein complex Ptf1, which is essential for fate determination and cell differentiation of pancreas. Recent studies have found that levels of Ptf1a in developing pancreas are closely related to the growth and differentiation of pancreatic cells, as well as the number of islet β cells. However, the regulatory mechanism of Ptf1a expression is still elusive. MicroRNAs are a class of approximately 22-nucleotide, non-coding RNAs, which regulate gene expression by cleaving target mRNAs or inhibiting their translation. In addition, some studies have indicated that miRNAs participate in regulating many processes in pancreatic development. Therefore, miRNAs may regulate the expression of Ptf1a in developing pancreas. In order to verify this hypothesis, four miRNAs which may regulate the expression of Ptf1a were firstly obtained by overlapping two prediction algorithms. Subsequently, by dual luciferase reporter system in vitro, three of the four miRNAs, miR-18a, miR-145 and miR-495, were proved to effectively inhibit Ptf1a expression, respectively, by binding to the 3′UTR of mouse Ptf1a mRNAs. Finally, expression patterns of miR-18a, miR-145 and miR-495 and Ptf1a were detected in the mouse pancreatic development via qRT-PCR and immunofluorescent staining. The results showed that the expression of miR-18a, miR-145 or miR- 495 is negatively correlated with that of Ptf1a mRNA and protein. Accordingly, these results indicated that miR-18a, miR-145 and miR-495 may regulate the expression of Ptf1a in developing mouse pancreas.