We have demonstrated that hairpin pyrrole (Py)-imidazole (Im) polyamide-CPI conjugates selectively induced luciferase gene silencing by sequence-specific alkylation of the coding region. Recently, we developed a new type of Py-Im polyamide CBI conjugate with an indole linker as a stable sequence-specific alkylating agent. In this study, we investigated the gene silencing ability of polyamides A, B and C, which potentially target specific sequences in the promoter region, non-coding strand, and coding strand of the green fluorescent protein (GFP) gene, respectively. The GFP vectors were transfected into human colon carcinoma cells (HCT116), and the cells treated with 100 nM of the polyamides for 24 h. Using direct observation of cell by fluorescence microscopy, a significant GFP-gene silencing effect was only seen with treatment with polyamide C. Polyamides A and B did not show such activity.