Objective To observe the effects of the Imiquimod solution on the mRNA expressions of cytokines(TNF-α, IL-1β and IL-6) and cytokine secretion from human epidermal Langerhans cells and human eternal cell lines—HaCaT keratinocyte to illuminate the immunoregulation mechanisms of Imiquimod. Methods Langerhans cells were sorted by density gradient centrifugation and magnetic technique. Langerhans cells and HaCaT cells were divided into experimental group and control group. The solution of 5 μg/ml Imiquimod was added to the experimental group. The total RNA and cell-free culture supernatants were harvested 4 hours after application. RT-PCR was conducted to detect the mRNA expressions of TNF-α, IL-1β and IL-6 in each group, cytokines secretion was determined by ELISA kits. Results Four hours after 5 μg/ml Imiquimod application, expression levels of TNF-α, IL-1β and IL-6 mRNA and cytokines secretion from Langerhans cells were statistically higher than those of controls(P<0.05). On the contrary, there was no statistically difference of the mRNA expression levels and cytokine secretion of TNF-α, IL-1β and IL-6 of HaCaT cells between the experimental group and the control group(P>0.05). Conclusion The solution of 5 μg/ml Imiquimod can increase the mRNA expression and secretion of TNF-α, IL-1β and IL-6 in sorted Langerhans cells. But in the HaCaT cells, Imiquimod fails to increase the mRNA expression and secretion of the above cytokines.