Na⁺/HCO₃^- cotransporter (NBC) mediates HCO₃^- import into the colonocyte via its pNBC1 isoform. While renal kNBC1 is inhibited by increased cAMP levels, we and others have recently shown structural and functional evidence for cAMP-dependent stimulation of pNBC1. Cholinergic stimulation activates renal NBC, but the effect on intestinal NBC is unknown. Therefore, crypts were isolated from murine proximal colon by Ca²⁺ chelation and loaded with the pH-sensitive dye BCECF. Na⁺/HCO₃^- cotransport activity was calculated from the di-methyl-amiloride-insensitive (DMA, 500 µM) pH_i recovery from an acid load in the presence of CO₂/HCO₃^- and the intracellular buffering capacity. Carbachol strongly increased Na⁺/HCO₃- cotransport activity compared to control rates. Calcium-chelation with BAPTA/AM, blockade of the M3 subtype of muscarinergic receptors with 4-DAMP, and inhibition of the Ca⁺⁺/calmodulin kinase isoform CaMKII with KN-62 all caused significant inhibition of the Carbachol-induced NBC activity increase. Furthermore, PKC inhibition with Goe6976 and Goe6850 significantly reduced the carbachol effect, which may be related to the unique N-terminal consensus site for PKC-dependent phosphorylation of pNBC1. We conclude that the Na⁺/HCO₃^- cotransporter in murine colon is thus activated by carbachol, consistent with its presumed function as an anion uptake pathway during intestinal anion secretion, but that the signal transduction pathways are distinct from those involved in cholinergic activation of the renal NBC1.