Poor chemotherapy efficacy in pancreatic cancer is attributed to limited drug permeation caused by the dense extracellular matrix (ECM) and drug degradation induced by tumor-colonizing bacteria. Here, a tumor-targeting probiotic-nanosystem is elaborately designed to remodulate ECM and selectively regulate tumor-colonizing bacteria for improving chemo-immunotherapy against pancreatic cancer. Specifically, drug-loaded liposomes are conjugated with Clostridium Butyricum (CB) via matrix metalloproteinase-2 (MMP-2)-responsive peptide to construct a probiotic-nanosystem. Particularly, vactosertib (VAC, a transforming growth factor-β1 receptor inhibitor) is delivered by probiotic-nanosystem to silence the active pancreatic stellate cells (PSCs) for inhibiting the development of ECM, resulting in a loosened ECM and providing a golden opportunity for the deep penetration of chemotherapy drugs and immune cells. Subsequently, gemcitabine (GEM) is efficiently delivered into the core of tumors via probiotic-nanosystem, achieving an enhanced chemotherapy efficacy. Noteworthily, CB can alleviate γ-proteobacteria-mediated GEM degradation through competitively reducing the contents of γ-proteobacteria and promoting the amounts of tumor-inhibiting bacteria, thereby significantly potentiating the therapeutic effect of GEM. The engineered probiotic-nanosystem can not only enhance the GEM-induced immunogenic cell death (ICD) of a pancreatic tumor to activate antitumor immune responses but also markedly increase the tumor-infiltration of effector immune cells to heighten tumoricidal immunity, offering a promising strategy for chemo-immunotherapy of pancreatic cancer.