DNA polymerase delta-interacting protein 38 (PDIP38) was identified in 2003 as a human DNA polymerase delta interacting protein which plays important roles in DNA repair, mitosis and vascular smooth muscle cells (VSMCs) migration. Our previous study showed that PDIP38 was expressed in mouse embryonic stem (ES) cells and upregulated in protein levels after differentiation from ES cells, while the expression in mRNA levels was not changed. We supposed that microRNA played key roles in the regulation of PDIP38 and the differentiation of ES cells. By bioinformatics assay, we predicted that PDIP38 was a potential target of microRNA--291a-5p (miR-291a-5p). Furthermore, we validated the possibility of miR-291a-5p to regulate the protein expression of PDIP38. Using luciferase reporter assay, realtime PCR and western blot methods, we firstly demonstrated that miR-291a-5p directly inhibited the expression of PDIP38. The present results shed a new light on the study of PDIP38 and miR-291a-5p in the differentiation of ES cells.