Imiquimod is an itch-promoting, small, synthetic compound that is generally used to treat genital warts and basal cell carcinoma. The pruritogenic effect of imiquimod is considered to be due to TLR7 activation; however that idea has been challenged by our studies showing intact pruritogenic effects of imiquimod in TLR7 KO mice. Thus, the signaling pathways of imiquimod have not been completely elucidated. Here we investigated the novel effects of imiquimod on intracellular calcium ([Ca(2+)]i) signaling. We found that imiquimod induces [Ca(2+)]i increases in PC12 and F11 cells, and even in NIH-3T3 and HEK293T cells, which do not express TLR7. This [Ca(2+)]i increase was due to Ca(2+) release from the internal store without extracellular Ca(2+) influx. Neither FCCP, a mitochondrial Ca(2+) reuptake inhibitor, nor dantrolene, a ryanodine receptor inhibitor, affected the imiquimod-induced [Ca(2+)]i increase. However, 2APB, an IP3 receptor blocker, inhibited the imiquimod-induced [Ca(2+)]i increase. U73122, a PLCβ inhibitor, failed to block the imiquimod-induced [Ca(2+)]i increase. These data indicate that imiquimod triggers IP3 receptor-dependent Ca(2+) signaling independently of TLR7.