The effect of inhibiting NO-synthase (NOS) or enhancing NO on the course of acute pancreatitis (AP) is controversial, in part because three NOS isoforms exist: neuronal- (nNOS), endothelial- (eNOS) and inducible- (iNOS). We investigated whether inhibition or selective gene deletion of NOS isoforms modified the initiation phase of caerulein-induced AP in mice and explored whether this affected pancreatic microvascular blood flow (PMBF). We investigated the effects of non-specific NOS inhibition with L-NNA (10 mg/kg IP) or targeted deletion of eNOS, nNOS or iNOS genes on the initiation phase of caerulein-induced AP in mice, using in vivo and in vitro models. Western blotting was performed to assess eNOS phosphorylation status, an indicator of enzyme activity, and microsphere studies were used to measure PMBF. L-NNA and eNOS deletion, but not nNOS or iNOS deletion, increased pancreatic trypsin activity and serum lipase during the initiation phase of in vivo caerulein-induced AP. L-NNA and eNOS did not affect trypsin activity in caerulein hyperstimulated isolated acini, suggesting that non-acinar events mediate the effect of NOS blockade in vivo. The initiation phase of AP in WT mice was associated with eNOS Thr-495 residue dephosphorylation, which accompanies eNOS activation, and a 178% increase in PMBF; these effects were absent in eNOS deleted mice. Thus eNOS is the main isoform influencing the initiation of caerulein-induced AP. eNOS derived NO exerts a protective effect through actions on non-acinar cell types, most likely endothelial cells to produce greater PMBF.