The aim of this study is to investigate whether BMP-2 regulates the oral sulcus formation of mouse embryonic tongue by modifying the expression of TIMP and MMP. The BMP-2 siRNA induced a 180% increase in the depth of oral sulcus cavity (P < 0.01) by stimulating the invagination of oral sulcus into the mesenchymal tissues consisting of tongue floor, whereas the recombinant BMP-2 suppressed the process in the organ culture system of mouse embryonic tongue. The BMP-2 siRNA induced a 60% decrease in the expression of TIMP-1 mRNA (P < 0.05) and a drastic decline in TIMP-1 protein was observed around the oral sulcus in the BMP-2 siRNA treated mandibles. The recombinant BMP-2 induced a 220% increases in the expression of TIMP-1 mRNA and the area of the immunostaining for TIMP-1 around the oral sulcus was larger in the mandibles treated with the recombinant BMP-2 than the vehicle. The BMP-2 siRNA induced a 60% increase in the expression of MMP-13 protein and a marked increase in the staining intensity for MMP-13 was observed in the epithelial region of the BMP-2 siRNA treated mandibles. The recombinant BMP-2 induced a 70% decrease in the expression of MMP-13 mRNA and the decrease was mainly observed in the tissues around oral sulcus. The expressions of BMP-2, TIMP-1, and MMP-13 were verified in the tissues around in vivo developing oral sulcus at E11, 12, and 13 by immunohistochemistry. These results suggest that BMP-2 regulates the formation of oral sulcus by altering the balance between TIMP-1 and MMP-13. Anat Rec 293:1408–1415, 2010. © 2010 Wiley-Liss, Inc.