Our previous study demonstrated that NYD-SP27 is a novel inhibitory PLC isoform expressed endogenously in human pancreas and upregulated in CFPAC-1 cells. The present study investigated the effect of NYD-SP27 down-regulation on the ATP-stimulated and Ca2+-dpendent pancreatic anion secretion by CFPAC-1 cell line using short-circuit current (I SC) recording. NYD-SP27 antisense-transfected CFPAC-1 (AT-CF) cells exhibited a significantly higher basal transmembrane potential difference and current than those of empty vector-transfected CFPAC-1 (VT-CF) cells. Cl− channel blocker, DPC or Glibenclamide (1 mM), and inhibitor of Na+-K+-Cl− cotransporter, bumetanide (100 μM) significantly inhibited the basal current in AT-CF cells. The inhibitor of adenylate cyclase, MDL12330A (20 μM), and Ca2+-dependent Cl− channel (CaCC) blocker, DIDS (100 μM) also significantly reduced the basal current in AT-CF. Apical application of ATP (10 μM) stimulated a fast transient I SC increase in VT-CF cells, but a more sustained rise with slower decline in AT-CF cells. Pretreatment with BAPTA-AM (50 μM) reduced the ATP-induced I SC response in AT-CF cells by 77.9%. PMA (1 μM), a PKC activator, inhibited the ATP-stimulated current increase (the transient peak) in VT-CF cells, but had no effect on the AT-CF cells. However, PKC inhibitor, staurosporine (40 μM) could inhibit the ATP-induced I SC response in AT-CF cells. The present results confirm the previously proposed inhibitory role of NYD-SP27 in the PLC pathway and demonstrate that the suppression of its expression could result in an enhancement of ATP-stimulated Ca2+ dependent pancreatic anion secretion.