Tooth bud is initiated by dental epithelium thickening and invaginated epithelium forms cap-like structure with condensed underlying mesenchyme. From this cap stage, specific epithelial cells form a cluster of non-dividing cells, primary enamel knot (PEK), and this PEK expresses abundant signaling molecules and functions as a signaling center for tooth development. Recently, hundreds of genes involved in tooth development have been reported by advanced genome-wide screening technology, but still remained unidentified genes obstruct elucidation of the precise molecular mechanisms underlying tooth development. In this work, we examined the expression patterns and developmental functions of a novel tooth germ-expressing gene, Dachshund1 (Dach1). Dach1, known as the cell fate regulator via controlling the transcription in various cell types, expressed in PEK region from cap stage to bell stage. In order to evaluate the developmental functions of Dach1 in tooth development, we cultivated the lower first molar at E12.5 for 2 and 4 days with or without treatments of antisense-oligodeoxynucleotides (AS-ODNs) against Dach1. After the knocking down of Dach1, morphological changes and cell physiology such as proliferation, cell death and migration patterns were examined. In addition, the expression levels of PEK-expressing genes such as Bmp2, Bmp7, Fgf4, Shh and Wnt5a were examined by using RT-qPCR and in situ hybridization methods. Based on these results, we suggest that Dach1 would involve in mice tooth morphogenesis through regulating the expressions of PEK related genes and cellular events to form the proper tooth structural formation.