Cryoablation, as a well-characterized technology, has multifarious clinical applications in solid malignancy. However, trans-biliary cryoablation for malignant biliary obstruction has not been reported yet. Thus, this study aimed to determine the efficacy and safety of trans-biliary cryoablation with a novel CO2 gas-based flexible cryoprobe in standardized preclinical settings. For fresh porcine liver ex vivo, the freezing efficacy of cryoablation was evaluated by using fresh porcine liver. The real-time CO2 flow rate, freezing temperature and freezing range were examined and the frozen appearance was visualized. In vivo study, acute and chronical effects were investigated by using the models of canine bile duct. Histopathology and laboratory examination were performed. The lowest temperature that the electrode could deliver to the tissue was -60.7 °C. At 60s after freezing, the tissue temperature dropped to -22.6 °C and -4.3 °C at 0.1 and 0.2 cm from the electrode center, respectively. The frozen size was greater in liver tissue ex vivo than that in bile duct tissue in vivo. No biliary hemorrhage, perforation, stricture, obstruction, and adjacent organ injury were observed. With histopathologic examination, acute intercellular vacuoles were observed in the lamina propria adjacent to the lumen. Chronic changes, including uneven coagulative necrosis, fibro-proliferation, inflammatory infiltration and connective tissue thickening were observed in the lamina propria of the all biliary samples. The results demonstrated CO2 gas-based trans-biliary cryoablation is safe and efficacious. These findings may provide a potential new modality for primary malignant biliary obstruction and malignant obstruction within a biliary stent and contribute to cryoablation of clinical practice.Copyright © 2022. Published by Elsevier Inc.