In the current study, we have characterized the global miRNA expression profile in mouse pancreatic acinar cells and during acute pancreatitis using next-generation RNA-Sequencing. We identified 324 known and 6 novel miRNAs being expressed in mouse pancreatic acinar cells. At basal state, miR-148a-3p, miR-375-3p, miR-217-5p, miR-200a-3p were among the most abundantly expressed whereas, miR-24-5p and miR-421-3p were least abundant. Treatment of acinar cells with caerulein (100 nM) and taurolithocholic acid 3-sulfate [TLC-S (250 μM)] induced numerous changes in miRNA expression profile. In particular, we found significant overexpression of miR-21-3p in acini treated with caerulein and TLC-S. We further looked at the expression of miR-21-3p in caerulein, L-arginine and caerulein + LPS induced acute pancreatitis mouse models and found 12, 21 and 50 fold increased expression in pancreas, respectively. In summary, this is the first comprehensive analysis of global miRNA expression profile of mouse pancreatic acinar cells in normal and disease conditions. Our analysis shows that miR-21-3p expression level correlates with severity of the disease.