The objective of this study was to explore the potential of CO₂ single contrast in-line phase contrast imaging (PCI) for pre-clinical small intestine investigation. The absorption and phase contrast images of CO₂ gas production were attained and compared. A further increase in image contrast was observed in PCI. Compared with CO₂-based absorption contrast imaging (ACI), CO₂-based PCI significantly enhanced the detection of mucosal microstructures, such as pits and folds. The CO₂-based PCI could provide sufficient image contrast for clearly showing the intestinal mucosa in living mice without using barium. We concluded that CO₂-based PCI might be a novel and promising imaging method for future studies of gastrointestinal disorders.