Accumulating evidence demonstrates that acetylcholine can directly modulate immune function in peripheral tissues including the spleen and gastrointestinal tract. However, the anatomical relationships between the peripheral cholinergic system and immune cells located in these lymphoid tissues remain unclear due to inherent technical difficulties with currently available neuroanatomical methods. In this study, mice with specific expression of the tdTomato fluorescent protein in choline acetyltransferase (ChAT)-expressing cells were used to label preganglionic and postganglionic cholinergic neurons and their projections to lymphoid tissues. Notably, our anatomical observations revealed an abundant innervation in the intestinal lamina propria of the entire gastrointestinal tract principally originating from cholinergic enteric neurons. The aforementioned innervation frequently approached macrophages, plasma cells, and lymphocytes located in the lamina propria and, to a lesser extent, lymphocytes in the interfollicular areas of Peyer's patches. In addition to the above innervation, we observed labeled epithelial cells in the gallbladder and lower intestines, as well as Microfold cells and T-cells within Peyer's patches. In contrast, we found only a sparse innervation in the spleen consisting of neuronal fibers of spinal origin present around arterioles and in lymphocyte-containing areas of the white pulp. Lastly, a small population of ChAT-expressing lymphocytes was identified in the spleen including both T- and B-cells. In summary, this study describes the variety of cholinergic neuronal and nonneuronal cells in a position to modulate gastrointestinal and splenic immunity in the mouse.