Ca2+/calmodulin-dependent protein (CaM) kinases play an important role in Ca2+-mediated secretory mechanisms. Previously, we demonstrated that a CaM kinase II inhibitor KN-62 had a small inhibitory effect on amylase secretion stimulated by CCK. In the present study, we investigated the effects of a myosin light chain kinase (MLCK) inhibitor on amylase secretion and Ca2+ signaling in rat pancreatic acini. A specific inhibitor of MLCK, wortmannin, inhibited amylase secretion stimulated by CCK-8 (30 pM) in a concentration-dependent manner. Wortmannin (10 μM) had no effects on basal secretion but reduced amylase secretion stimulated by CCK-8 (30 pM) by 67 ± 3%. Wortmannin inhibited amylase secretion stimulated by calcium ionophore (A23187) and phorbol ester (TPA). Wortmannin also inhibited amylase response to thapsigargin by 76 ± 8% and to both thapsigargin and TPA by 52 ± 10%. Ca2+ oscillations evoked by CCK-8 (10 pM) were inhibited by wortmannin (10 μM). Wortmannin had a little inhibitory effect on an initial rise in [Ca2+]i, and abolished a subsequent sustained elevation of [Ca2+]i evoked by 1 nM CCK-8. In conclusion, MLCK plays a crucial role in amylase secretion from pancreatic acini and regulates Ca2+ entry from the extracellular space.