Background: Cholera remains a major global health problem, causing high output diarrhea leading to severe dehydration and shock in developing countries. We aimed to determine whether vasoactive intestinal polypeptide (VIP), the mediator of pancreatic cholera syndrome, has a role in the pathophysiology of human cholera. Methods: We conducted a prospective observational study of cholera cases hospitalized with severe dehydration. Plasma and stool water levels of VIP were measured just after admission, after complete rehydration (3–4 h), at 24 h post-rehydration and at discharge after diarrhea ceased. Results: In total, 23 cholera patients were examined between January and August 2018. The geometric mean of stool VIP (sVIP) and plasma VIP (pVIP) on admission were 207.67 and 8.34 pmol/L, respectively. pVIP values were all within the normal range (</= 30 pcmol/L); however, sVIP levels were very high at all timepoints, though less so just after rehydration. In multivariable GEE models, after adjustment for covariates, sVIP levels were significantly associated with duration of hospitalization (p = 0.026), total stool volume (p = 0.023) as well as stool output in the first 24 h (p = 0.013). Conclusions: The data suggest that VIP, which is released by intestinal nerves, may play an important role in human choleragenesis, and inhibitors of intestinal VIP merit testing for potential therapeutic benefits.