Interaction of the neuropeptide substance P (SP) with its high affinity neurokinin1 receptor (NK1R) plays an important role in the pathophysiology of acute pancreatitis. SP is known to stimulate production of chemokines MCP-1, MIP-1and MIP-2 in pancreatic acinar cells via activation of nuclear factor (NF)B. However, the signaling mechanisms by which SPNK1R interaction induces NFB activation and chemokine production remain unclear. To that end, in this study we investigated the participation of protein kinase C (PKC) in SP-induced chemokine production in pancreatic acinar cells. In this study we showed that SP stimulated an early phosphorylation of the PKC isoform PKC, followed by an increased activation in MAPKK kinase MEKK1, MAP kinases ERK and JNK as well as transcription factors, NFB and AP-1, driven chemokine production. Depletion of PKC with the specific PKC inhibitor, rottlerin dose dependently decreased SP-induced PKC, MEKK1, ERK, JNK, NFB and AP-1 activation. Moreover, rottlerin inhibited SP-induced chemokine production in a concentration dependent manner both at mRNA and protein levels. We also demonstrated that PKC activation was attenuated by CP96345, a selective NK1R antagonist, thus showing that PKC activation was indeed mediated by SP in pancreatic acinar cells. These results show that PKC is an important proinflammatory signal transducer for SPNK1R-induced chemokine production in pancreatic acinar cells.