ABSTRACT Peptide backbone cyclization is a widely used approach to improve the activity and stability of small peptides but until recently it had not been applied to peptides with multiple disulfide bonds. Conotoxins are disulfide‐rich conopeptides derived from the venoms of cone snails that have applications in drug design and development. However, because of their peptidic nature, they can suffer from poor bioavailability and poor stability in vivo. In this study two P‐superfamily conotoxins, gm9a and bru9a, were backbone cyclized by joining the N‐ and C‐termini with short peptide linkers using intramolecular native chemical ligation chemistry. The cyclized derivatives had conformations similar to the native peptides showing that backbone cyclization can be applied to three disulfide‐bonded peptides with cystine knot motifs. Cyclic gm9a was more potent at high voltage‐activated (HVA) calcium channels than its acyclic counterpart, highlighting the value of this approach in developing active and stable conotoxins containing cyclic cystine knot motifs. © 2015 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 104: 682–692, 2015.