Within the brain, the satiety peptide GLP-1 is produced by preproglucagon (PPG) neurons, located primarily in the nucleus tractus solitarius. PPG axons are widely distributed throughout brain, mainly to autonomic control areas1, 2. The PPG innervation pattern corresponds well with regional GLP-1 receptor expression3. GLP-1 receptor mRNA is also found throughout the rat spinal cord3; but to date, nothing is known about the spinal distribution of PPG axons. Here, we analysed the distribution of PPG promoter-driven YFP expression in coronal and longitudinal sections of seven mouse spinal cords between thoracic segment 1 (T1) and sacral segment 3 (S3). We also explored whether PPG axons form close appositions with cholinergic neurons; including preganglionic autonomic neurons and somatic motor neurons. YFP and choline acetyltransferase (ChAT) immunoreactivity was visualised with two colour immunoperoxidase labelling using black (YFP) and brown (ChAT) reaction products. Many YFP-immunoreactive (IR) non-varicose axons travelled rostrocaudally in white matter tracts. The greatest density of these axons occurred in the ventral white commissure and around the ventral median fissure. They were also distributed throughout the lateral and ventral funiculi in rostral thoracic segments. Their density declined from rostral to caudal segments. In spinal segments T1-L2, where sympathetic preganglionic neurons are located, many varicose, YFP-IR axons travelled between the intermediolateral cell column and the intercalated nucleus. Many of these axons closely apposed ChAT-IR cell bodies and proximal dendrites. There were also many varicose mediolaterally-oriented axons containing YFP-IR in lamina X; these formed close appositions on ChAT-positive lamina X perikarya. In segments S1 and S2, rare YFP-IR terminals closely apposed ChAT-IR somata in the parasympathetic nucleus, which contains the parasympathetic preganglionic neurons. Varicose YFP-IR axons projected ventrolaterally through lamina VII into laminae VIII and IX; areas containing limb motor neurons. Here, close appositions between YFP-IR axons and ChAT-IR motor neurons were infrequent. A few dorsolaterally-oriented axons occurred in lamina V. No YFP-IR fibres occurred more dorsally. In addition to YFP-positive axons, a small numbers of YFP-IR cell bodies were present in lower lumbar and upper sacral segments, mostly within laminae V and VI. These results demonstrate that PPG neurons innervate cholinergic neurons in both autonomic and somatic motor regions of the spinal cord, with sympathetic areas receiving the densest innervation. Like in other parts of the CNS, there is a good correlation between the distribution of spinal GLP-1 receptors and the distribution of spinal PPG axons. These observations indicate that PPG neurones could directly modulate sympathetic outflow through their inputs to sympathetic preganglionic neurons.