Neuropeptide modulation of immune cell function is an important mechanism of neuro-immune intersystem crosstalk. Substance P (SP) is one such key neuropeptide involved. In this study, we investigated the yet unexplored cellular mechanisms of SP-mediated inflammatory responses in macrophages using a mouse macrophage-like cell line RAW 264.7 and isolated peritoneal macrophages. We found that the conventional PKC and novel PKC and were selectively activated by SP via its primary neurokinin-1 receptor (NK-1R) on the cells. Activation of these PKC isoforms mediated the activation of downstream extracellular signal-regulated kinase-1/2 (ERK1/2) and the transcription factor NF-B, which drove the transcription of inducible chemokines in macrophages. Additionally, phosphoinositide 3-kinase (PI3K)-Akt was also activated by SP/NK-1R in macrophages. Inhibition of PI3K-Akt pathway attenuated ERK1/2 and NF-B activation, suggesting it also played a part in SP-induced cellular inflammatory response. Kinetic analysis indicated that PKC isoforms induced early ERK1/2 activation, while PI3K-Akt contributed to the pathway at later time points. It was further demonstrated that PKC and PI3K-Akt were activated independent of each other. Collectively, our results suggest that SP/NK-1R activates two convergent proinflammatory signaling pathways, PKCs and PI3K-Akt, resulting in ERK1/2 and NF-B activation and chemokine production in mouse macrophages.—Sun, J., Ramnath, R. D., Tamizhselvi, R., Bhatia, M. Role of protein kinase C and phosphoinositide 3-kinase-Akt in substance P-induced proinflammatory pathways in mouse macrophages.