Odontoblasts are terminally differentiated cells that play a vital role in dentinogenesis. The differentiation of odontoblasts is regulated by a variety of genetic and epigenetic mechanisms. Our previous microRNA microarray studies verified that miR-338-3p was up-regulated during odontoblast differentiation. The purpose of this study was to determine the function of miR-338-3p during odontoblast differentiation. The upregulation of miR-338-3p expression during odontoblast differentiation was validated by qRT-PCR. Odontoblast differentiation was enhanced after over-expression of miR-338-3p, while a loss of function approach using a miR-338-3p inhibitor impaired odontoblast differentiation. Bioinformatic analysis identified Runx2 as a potential target of miR-338-3p. Overexpression of miR-338-3p caused a decreased in the expression of Runx2 at both mRNA and protein levels, while Runx2 expression increased after treatment with miR-338-3p inhibitors. Furthermore, the activity of a luciferase reporter plasmid containing the 3'-UTR of Runx2 was significantly suppressed by ectopic expression of miR-338-3p. These results suggested that miR-338-3p promotes odontoblast differentiation through targeting Runx2.