Pancreatitis‐associated protein (PAP) is a secretory protein not normally expressed in healthy pancreas but highly induced during acute pancreatitis. While PAP has been shown to be anti‐bacterial and anti‐apoptotic in vitro, its definitive biological function in vivo is not clear. To elucidate the function of PAP, antisense oligodeoxyribonucleotides (AS‐PAP) targeting all three isoforms of PAP were administered via intrapancreatic injections (5 mg kg ⁻¹ day ⁻¹ , 2 days) to rats prior to induction of pancreatitis. Severity of pancreatitis and cytokine gene expression in peripheral blood mononuclear cells (PBMC) were evaluated. Administration of AS‐PAP, but not the scrambled oligodeoxyribonucleotide (SC‐PAP) control, reduced pancreatitis‐induced PAP expression by 55.2 ± 6.4%, 44.0 ± 8.9%, and 38.9 ± 10.7% for PAP isoforms I, II, and III, respectively, compared to saline‐treated controls (P < 0.05 for all). Inhibition of PAP expression significantly worsened pancreatitis: serum amylase activity, pancreas wet weight (reflecting edema), and serum C‐reactive protein levels all increased in AS‐PAP‐treated animals compared to SC‐PAP‐treated controls (by 3.5‐, 1.7‐, and 1.7‐fold, respectively; P < 0.05 for all). Histopathologic evaluation of pancreas revealed worsened edema, elevated leukocyte infiltration, and fat necrosis after AS‐PAP treatment. Gene expressions of IL‐1 μm and IL‐4 were significantly higher in PBMC isolated from AS‐PAP‐treated rats compared to SC‐PAP controls. This is the first in vivo evidence indicating that PAP mediates significant protection against pancreatic injury. Our data suggest that PAP may exert its protective function by suppressing local pancreatic as well as systemic inflammation during acute pancreatitis.