Myristoylated alanine-rich protein kinase C substrate (MARCKS) is a cellular substrate for protein kinase C (PKC). Recently, we have shown that PKC isoforms-α and -δ, as well as the Rho/Rho kinase (ROK) pathway, play a role in phorbol 12-myristate 13-acetate (PMA)-mediated secretion of the gut peptide neurotensin (NT) in the BON human endocrine cell line. Here, we demonstrate that activation of MARCKS protein is important for PMA- and bombesin (BBS)-mediated NT secretion in BON cells. Small interfering RNA (siRNA) to MARCKS significantly inhibited, whereas overexpression of wild-type MARCKS significantly increased PMA-mediated NT secretion. Endogenous MARCKS and green fluorescent protein-tagged wild-type MARCKS were translocated from membrane to cytosol upon PMA treatment, further confirming MARCKS activation. MARCKS phosphorylation was inhibited by PKC-δ siRNA, ROKα siRNA, and C3 toxin (a Rho protein inhibitor), suggesting that the PKC-δ and the Rho/ROK pathways are necessary for MARCKS activation. The phosphorylation of PKC-δ was inhibited by C3 toxin, demonstrating that the role of MARCKS in NT secretion was regulated by PKC-δ downstream of the Rho/ROK pathway. BON cell clones stably transfected with the receptor for gastrin releasing peptide, a physiologic stimulant of NT, and treated with BBS, the amphibian equivalent of gastrin releasing peptide, demonstrated a similar MARCKS phosphorylation as noted with PMA. BBS-mediated NT secretion was attenuated by MARCKS siRNA. Collectively, these findings provide evidence for novel signaling pathways, including the sequential regulation of MARCKS activity by Rho/ROK and PKC-δ proteins, in stimulated gut peptide secretion.