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Given that no studies have reported the use of adipose-derived stem cell sheets for the prevention of pancreatic fistulas, it is unclear whether adipose-derived stem cell sheets are effective at preventing this complication. The aim of this study was to evaluate the efficacy of novel therapy for the prevention of pancreatic fistulas using adipose-derived stem cell sheets treated with mannose.The rat pancreatic duct (splenic duct) and surrounding pancreatic parenchyma were transected to induce a pancreatic fistula. Adipose-derived stem cell sheets with or without mannose treatment were attached to the pancreatic transection stump. Amylase and lipase levels were measured in both the ascites and serum. The expression of 40 cytokines in human adipose-derived stem cells with and without mannose treatment was investigated using a multiplex assay.The adipose-derived stem cell sheets remained at the initial attachment site at 48 hours after operation. Macroscopically, more severe degeneration and adhesion in the peritoneal cavity were observed in the untreated rats than in the rats treated with adipose-derived stem cell sheets. The levels of ascitic amylase in the untreated, adipose-derived stem cell-sheet-treated, and adipose-derived stem cell-sheet-with-mannose-treated rats were 10.7 ± 2.9 × 104 U/L, 2.6 ± 0.9 × 104 U/L, and 1.5 ± 0.3 × 104 U/L, respectively. The levels of ascitic lipase in the untreated, adipose-derived stem cell-sheet-treated and adipose-derived stem cell-sheet-with-mannose-treated rats were 9.5 ± 2.9 × 103 U/L, 4.0 ± 3.3 × 103 U/L, and 0.4 ± 0.2 × 103 U/L, respectively. Significant differences were found in both the ascitic and serum levels of amylase and lipase between the untreated rats and the rats treated with adipose-derived stem cell sheets with mannose (P < .05). Fibroblast growth factor 2 gene expression levels were greater in human adipose-derived stem cells treated with mannose than in human adipose-derived stem cells treated without mannose.Adipose-derived stem cell sheets treated with mannose are effective for preventing pancreatic fistulas and have promising potential for clinical applications. The enhancing effect of mannose on the function of adipose-derived stem cells may be partially explained by induction of fibroblast growth factor 2.