The activation of Wingless-type (Wnt)/β-catenin signaling is of fundamental importance in organogenesis. Wnt signaling is also known to regulate signaling crucial for tooth development. However, the underlying mechanism of Wnt activation or inhibition remains largely unknown. Here, we demonstrate that Wnt inhibitory factor 1 (Wif1), a secreted Wnt antagonist, localizes to the dental epithelium and mesenchyme during early tooth development. Specifically, Wif1 is strictly expressed in the enamel knot at embryonic day 14.5 (E14.5) and E16.5. The functional significance of Wif1 during tooth morphogenesis remains to be clarified. Our results reveal that the promotion of apoptosis by the knockdown of Wif1 leads to a delay in an early event during tooth development. This study therefore provides novel insights into the role of Wif1 and validates Wif1 as a potent target in WNT signaling during tooth development. We suggest that the enamel knots are central regulators of tooth development. Furthermore, Wif1 localizes to the enamel knot in which Wif1 regulates apoptosis by mediating and regulating Wnt-β-catenin signaling. Thus, Wif1 plays an essential role in tooth development.