This work describes the synthesis and activity of a novel backbone cyclic (BC) peptide library based on the sequence of the HIV-1 Rev arginine-rich motif (ARM). All the peptides in the library possess the same sequence but differ in their ring-moiety properties. The BC peptides were synthesized using simultaneous multiple-peptide synthesis and were fully assembled using bis(trichloromethyl)carbonate as a coupling agent. All the peptides in the library had inhibitory effects on the binding of Rev-GFP to importin β in vitro. Studies performed with one of the BC Rev-ARM analogues, Rev-13, demonstrated that, like its parental linear peptide, it is karyophilic; i.e., it is able to mediate the nuclear import of conjugated bovine serum albumin (BSA) molecules. The cell penetrating properties of the BC peptides were assessed utilizing an ELISA-based system. This assay provides a quantitative evaluation of cell penetration. Most of the peptides from the library were able to penetrate intact Colo-205 cells to varying degrees. Furthermore, these BC peptides were able to carry BSA into intact Colo-205 cells. In addition to its cell penetrating and binding properties, the BC Rev-13 analogue inhibited Rev-induced gene expression in HeLa cells by 60−70% in the low micromolar range and exhibited no cell toxicity. The potential of BC peptides bearing ARM domains as lead compounds for the production of anti-HIV drugs is discussed.