Introduction: Pancreatic ductal adenocarcinoma (PDAC) is a rapidly increasing cause of mortality whose dismal prognosis is mainly due to overwhelming chemoresistance. New therapeutic approaches include physical agents such as ultrasonic cavitation, but clinical applications require further insights in the mechanisms of cytotoxicity. Three dimensional in vitro culture models such as spheroids exploit realistic spatial, biochemical and cellular heterogeneity that may bridge some of the experimental gap between conventional in vitro and in vivo experiments. Purpose: to assess the feasibility and efficiency of inertial cavitation associated or not with chemotherapy, in a spheroid model of PDAC. Methods: we used DT66066 cells ,derived from a genetically-engineered murine PDAC, isolated from KPC-transgenic mice (LSL-KrasG12D/+; LSL-Trp53R172H/+; Pdx-1- Cre). Spheroids were obtained by either a standard centrifugation-based method, or by using a magnetic nano-shuttle method allowing the formation of spheroids within 24 hours and facilitating their handling. The spheroids were exposed to ultrasonic inertial cavitation in a specially designed setup. Four conditions were studied: control, gemcitabine alone , US cavitation alone , US cavitation + gemcitabine. Five US inertial cavitation indexes, corresponding to increased US intensities, were evaluated . The effectiveness of treatment was assessed after 24 hours with the following criteria: spheroid size (growth), ratio of phase S cells (proliferation), proportion of cells in apoptosis or necrosis (cellular mortality). These parameters were assessed by quantitative immunofluorescence techniques. Results: The 3D culture model presented excellent reproducibility. Eight or nine spheroids were analyzed for each condition. Cavitation induced a significant decrease in the size of spheroids , an effect significantly correlated to an increasing cavitation index (p < 0.0001). The treatment induced cell death whose predominant mechanism was necrosis (p < 0.0001). There was a tendency to a synergistic effect of US cavitation and gemcitabine at 5μM concentration, however significant in only one of the cavitation indexes used (p = 0. 013). Conclusion: Ultrasonic inertial cavitation induced a significant reduction of tumor growth in a spheroid model of PDAC., with necrosis rather than apoptosis as a Cell dominant mechanism of cell death. More investigations are needed to understand the potential role of inertial cavitation in overcoming chemoresistance.