Plant cyclotides are a large family of naturally occurring circular proteins that are produced from linear precursors containing one, two or three cyclotide domains. The mechanism of excision of the cyclotide domains and ligation of the free N- and C-termini to produce the circular peptides has not been elucidated. Here, we investigate production of the prototypic cyclotide kalata B1 from the precursor Oak1 from the African plant Oldenlandia affinis. Immunoprecipitation experiments and MALDI-TOF mass spectrometry analysis showed that O. affinis only produces mature kalata B1, whereas transgenic Arabidopsis thaliana, Nicotiana tabacum and Nicotiana benthamiana produced both linear and circular forms. Circular peptides were not produced when a highly conserved asparagine residue at the C-terminal processing site of the cyclotide domain was replaced with an alanine or an aspartate residue, or when the conserved C-terminal tripeptide motif was truncated. We propose that there are two processing pathways in planta: one to produce the mature cyclotide and the other to produce linear variants that ultimately cannot be cyclized.