Unlike other types of T helper (Th) responses, whether the development of Th2 cells requires instruction from particular subset of dendritic cells (DCs) remains unclear. By using an in vivo depletion approach, we have shown that DCs expressing CD301b were required for the generation of Th2 cells after subcutaneous immunization with ovalbumin (OVA) along with papain or alum. CD301b+ DCs are distinct from epidermal or CD207+ dermal DCs (DDCs) and were responsible for transporting antigen injected subcutaneously with Th2-type adjuvants. Transient depletion of CD301b+ DCs resulted in less effective accumulation and decreased expression of CD69 by polyclonal CD4+ T cells in the lymph node. Moreover, despite intact cell division and interferon-γ production, CD301b+ DC depletion led to blunted interleukin-4 production by OVA-specific OT-II transgenic CD4+ T cells and significantly impaired Th2 cell development upon infection with Nippostrongylus brasiliensis. These results reveal CD301b+ DDCs as the key mediators of Th2 immunity.