Cyclotides are plant-derived disulfide-rich peptides comprising a cyclic cystine knot, which confers remarkable stability against thermal, proteolytic, and chemical degradation. They represent an emerging class of G protein-coupled receptor (GPCR) ligands. In this study, utilizing a screening approach of plant extracts and pharmacological analysis we identified cyclotides from Carapichea ipecacuanha to be ligands of the κ-opioid receptor (KOR), an attractive target for developing analgesics with reduced side effects and therapeutics for multiple sclerosis (MS). This prompted us to verify whether [T20K]kalata B1, a cyclotide in clinical development for the treatment of MS, is able to modulate KOR signaling. T20K bound to and fully activated KOR in the low μM range. We then explored the ability of T20K to allosterically modulate KOR. Co-incubation of T20K with KOR ligands resulted in positive allosteric modulation in functional cAMP assays by altering either the efficacy of dynorphin A1-13 or the potency and efficacy of U50,488 (a selective KOR agonist), respectively. In addition, T20K increased the basal response upon cotreatment with U50,488. In the bioluminescence resonance energy transfer assay T20K negatively modulated the efficacy of U50,488. This study identifies cyclotides capable of modulating KOR and highlights the potential of plant-derived peptides as an opportunity to develop cyclotide-based KOR modulators.