Gulf War Illness (GWI) results from chemical exposure during the Gulf War, with notable impacts on gastrointestinal motility. Due to the limited demographic impacted by this ailment, an in‐depth investigation of the GWI has yielded little regarding the underlying pathophysiological mechanisms. Here, the hypothesis that exposure to pyridostigmine bromide (PB) results in severe enteric neuro‐inflammation, that cascades to disruptions in colonic motility, is tested. The analyses are performed on male C57BL/6 mice that are treated with physiologically similar doses of PB given to GW veterans. When colonic motility is assessed, GWI colons have significantly reduced forces in response to acetylcholine or electrical field stimulation. GWI is also accompanied by high levels of pro‐inflammatory cytokines and chemokines, associated with increased numbers of CD40+ pro‐inflammatory macrophages within the myenteric plexus. Enteric neurons responsible for mediating colonic motility reside within the myenteric plexus, and PB exposure reduced their numbers. Significant smooth muscle hypertrophy is also observed due to increased inflammation. Together, the results show that PB exposure caused functional and anatomical dysfunction, promoting impaired motility within the colon. Achieving a greater understanding of the mechanisms of GWI will allow more refinement in therapeutic options that improve veterans’ quality of life.