Key points Submucosal neurons are crucial regulators of gut secretion. Despite significant interest in using mouse models for enteric neuropathies, much is still unknown about their submucous innervation. We examined properties of submucosal neurons in the mouse distal colon using immunohistochemical and intracellular recording techniques, and investigated colonic regional differences in neurochemistry and neurally mediated ion transport responses. Two main neurochemical but not electrophysiological classes of neurons were identified: cholinergic (containing choline acetyltransferase) and non‐cholinergic. Non‐cholinergic neurons had one or two axons; the cholinergic neurons examined were uniaxonal. Neurons exhibited predominantly nicotinic fast excitatory postsynaptic potentials and somatic action potentials mediated by tetrodotoxin‐resistant voltage‐gated channels. The distal colon had smaller ganglia, a higher proportion of cholinergic neurons (they remain a minority) and a larger nicotinic secretory component than the proximal colon. Properties of submucosal neurons in the mouse distal colon differ from other colonic regions, and from submucosal neurons in other species. Abstract Submucosal neurons are vital regulators of water and electrolyte secretion and local blood flow in the gut. Due to the availability of transgenic models for enteric neuropathies, the mouse has emerged as the research model of choice, but much is still unknown about the murine submucosal plexus. The progeny of choline acetyltransferase (ChAT)‐Cre × ROSA26YFP reporter mice, ChAT‐Cre;R26R‐yellow fluorescent protein (YFP) mice, express YFP in every neuron that has ever expressed ChAT. With the aid of the robust YFP staining in these mice, we correlated the neurochemistry, morphology and electrophysiology of submucosal neurons in distal colon. We also examined whether there are differences in neurochemistry along the colon and in neurally mediated vectorial ion transport between the proximal and distal colon. All YFP+ submucosal neurons also contained ChAT. Two main neurochemical but not electrophysiological groups of neurons were identified: cholinergic (containing ChAT) or non‐cholinergic. The vast majority of neurons in the middle and distal colon were non‐cholinergic but contained vasoactive intestinal peptide. In the distal colon, non‐cholinergic neurons had one or two axons, whereas the cholinergic neurons examined had only one axon. All submucosal neurons exhibited S‐type electrophysiology, shown by the lack of long after‐hyperpolarizing potentials following their action potentials and fast excitatory postsynaptic potentials (EPSPs). Fast EPSPs were predominantly nicotinic, and somatic action potentials were mediated by tetrodotoxin‐resistant voltage‐gated channels. The size of submucosal ganglia decreased but the proportion of cholinergic neurons increased distally along the colon. The distal colon had a significantly larger nicotinic ion transport response than the proximal colon. This work shows that the properties of murine submucosal neurons and their control of epithelial ion transport differ between colonic regions. There are several key differences between the murine submucous plexus and that of other animals, including a lack of conventional intrinsic sensory neurons, which suggests there is an incomplete neuronal circuitry within the murine submucous plexus.