Imiquimod (Imiq) is a synthetic imizoquinoline compound which can act on Toll-like receptor (TLR)7 and transduce signals involved in cell activation. We investigated the role of Imiq on contact hypersensitivity (CHS) and explored the potential mechanisms of mast cells involved in the process. Topical application of Imiq cream augmented DNFB mediated CHS in C57BL/6 mice. Imiq application induced skin inflammation and increased the number of dendritic cells (DCs) in the draining lymph nodes (DLNs). The splenic cell proliferation to DNBS in DNFB and Imiq treated mice was greater than that in mice of DNFB treatment alone. Peritoneal cell-derived mast cells (PCMCs) expressed TLR7 mRNA. The results from toluidine blue staining for mast cells and histamine detection indicated that Imiq alone did not induce mast cell degranulation while Imiq plus DNFB significantly induced mast cell degranulation. Cromolyn, pyrilamine and cimetidine attenuated CHS reaction induced by Imiq. Our findings suggest that Imiq could augment the intensity of CHS reaction. The mechanisms underlying the effect may relate to histamine release by mast cells and induction of DC homing to DLNs. Blocking histamine action in early time of allergen contact is beneficial to the alleviation of CHS.