Suppressive potencies of tetrandrine, isotetrandrine, fangchinoline, berbamine, dauricine, cepharanthine, and armepavine on expression and activation of NF-κB in MOLT-4 cells, MOLT-4/DNR cells, peripheral blood mononuclear cells (PBMCs) of healthy subjects and PBMCs of dialysis patients were compared. In MOLT-4 cells, the suppressive potencies evaluated by the IC50 values were isotetrandrine > cepharanthine > tetrandrine > dauricine > fangchinoline > berbamine or armepavine. In MOLT-4/DNR cells, the order was isotetrandrine > tetrandrine > cepharanthine > fangchinoline > dauricine > berbamine or armepavine. In PBMCs of healthy subjects, the order was isotetrandrine > dauricine > fangchinoline > tetrandrine > cepharanthine > berbamine or armepavine. In PBMCs of dialysis patients, the order was isotetrandrine > fangchinoline > tetrandrine > cepharanthine > dauricine > berbamine or armepavine. Among them, isotetrandrine showed the strongest inhibitory effects on the expression of NF-κB and p-NF-κB in these cells. Accordingly, both 7- and 12-substitutions are suggested to influence the suppressive potency of bisbenzylisoquinoline alkaloids, though 7-substitution is likely to have less contribution. Bisbenzylisoquinoline alkaloid seems to be more suitable than monobenzylisoquinoline alkaloid to serve as a lead compound. Bisbenzylisoquinoline alkaloids with an 18-membered ring formed by two oxygen bridges seem to be superior to those with one oxygen. However, the binding sites of two oxygen bridges on the phenyl ring appear to have limited influence. These findings provide further insight into structure–activity relationships to the development of active analogs of this promising class of drugs for the treatment of diseases mediated by abnormalities of T cell activation.