Cyclotides are disulfide-rich macrocyclic peptides that display a wide range of bioactivities and represent an important group of plant defense peptide biologics. A few linear variants of cyclotides have recently been identified. They share a high sequence homology with cyclotides but are biosynthetically unable to cyclize from their precursors. All hitherto reported cyclotides and their acyclic variants were isolated from dicot plants of the Rubiaceae, Violaceae, Cucurbitaceae, and recently the Fabaceae and Solanaceae families. Although several cyclotide-like genes in the Poaceae family were known from the data mining of the National Center for Biotechnology Information (NCBI) nucleotide database, their expression at the protein level has yet to be proven. Here, we report the discovery and characterization of nine novel linear cyclotides, designated as panitides L1–9, from the Panicum laxum of the Poaceae family and provide the first evidence of linear cyclotides at the protein level in a monocot plant. Disulfide mapping of panitide L3 showed that it possesses a cystine knot arrangement similar to cyclotides. Several panitides were shown to be active against Escherichia coli and cytotoxic to HeLa cells. They also displayed a high stability against heat and proteolytic degradation. Oxidative folding of the disulfide-reduced panitide L1 showed that it can fold efficiently into its native form. The presence of linear cyclotides in both dicots and monocots suggests their ancient origin and existence before the divergence of these two groups of flowering plants. Moreover, the Poaceae family contains many important food crops, and our discovery may open up new avenues of research using cyclotides and their acyclic variants in crop protection.