Dengue virus (DENV) causes about 50-100 million cases per year worldwide. However, there is still a big challenge in developing antiviral drugs against DENV infection. Some derivatives of alkaloid (-)-cytisine, like other alkaloid analogs, have been proposed for their antiviral potential. This study investigated antiviral activity and mechanisms of the cytisine derivatives, and discovered the structure-activity relationship against DENV. The antiviral assays were performed using one strain of DENV1 and DENV2, and two cell lines Vero E6 and A549. The structure-activity relationship of the effective compounds was also evaluated using combination of time-of-addition/removal assay and molecular docking. Compounds 3, 4, 12 (N-allylcytisine-3-thiocarbamide), 16, and 20 exhibited the high antiviral activity with IC(50) values of lower than 3 muM against DENV1 and DENV2. Of them, the derivative 12 showed the highest antiviral activities against DENV1 (IC(50) = 0.14 muM) and DENV-2 (IC(50) = <0.1 muM), exhibiting the potent inhibition on virus attachment and entry stages. Meanwhile, the compounds 4 and 20 had a strong inhibition at the post-entry stage (IC(50) = <0.1 muM). A correlation between the experimental pIC(50) values and predicted pKi calculated by docking of compounds into DENV E protein was significant, correlating with the impact of compound 12 on the attachment stage, but compounds 4, and 20 on post-entry stage. The results provided the insight into the directions of synthetic modifications of starting (-)-cytisine as the inhibitors of DENV E protein at attachment and entry stages of DENV life cycle. CI - Copyright (c) 2021 Elsevier Ltd. All rights reserved.