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The aim of the study was to investigate the biochemical and functional properties of a rat acellular pancreatic bioscaffolds (APBs).Fresh pancreata from 10 rats were soaked and perfused through portal veins using Easy-Load Digital Drive peristaltic pumps. The histological structure, extracellular matrix composition, and the DNA content of the APBs were evaluated. Biocompatibility studies had also been performed. The proliferation and differentiation of AR42J pancreatic acinar cells were assessed.The pancreatic tissue became translucent after decellularization. There were no visible vascular endothelial cells, cellular components, or cracked cellular debris. The extracellular matrix components were not decreased after decellularization (P > 0.05); however, the DNA content was decreased significantly (P < 0.05). The subcutaneous implantation sites showed low immunological response and low cytotoxicity around the APB. The proliferation rate was higher and the apoptosis rate was lower when AR42J cells were cultured on APB (P < 0.05). The gene expression and the protein expression were higher for the APB group (P < 0.001).Our findings support the biological utility of whole pancreas APBs as biomaterial scaffolds, which provides an improved approach for regenerative medicine.