Plant-based human vaccines have been actively developed in recent years, and rice (Oryza sativa L.) is one of the best candidate crops for their production and delivery. By expressing a modified cholera toxin B (CTB) subunit, we previously developed MucoRice-CTB, a rice-based vaccine against cholera, which is caused by infection of the intestine with the bacteria Vibrio cholerae. MucoRice-CTB lines have been extensively characterized by whole-genome sequencing and proteome analyses to evaluate the mutation profiles and proteome status, respectively. Here, we report non-targeted metabolomic profiling of the MucoRice-CTB transgenic rice line 51A (MR-CTB51A), MucoRice-RNAi (MR-RNAi), and their non-transgenic parent line by using gas chromatography-time-of-flight mass spectrometry. The levels of several amino acids, organic acids, carbohydrates, lipids, and secondary metabolites were significantly increased in MR-CTB51A compared with the non-transgenic parent line. These metabolomics results complement essential information obtained by genome sequencing and proteomics approaches, thereby contributing to comprehensive understanding of the properties of MucoRice-CTB as a plant-based vaccine.