The mechanisms underlying gut development, especially peristalsis, are widely studied topics. However, the causes of gut peristalsis-related diseases, especially Opioid-Induced Bowel Dysfunction (OIBD) disorder, have not been well defined. Therefore, our study used zebrafish, a popular model for studying both gut development and peristalsis, and DCFH-DA, a dye that clearly labels the live fish gut lumen, to characterize the formation process of gut lumen as well as the gut movement style in vivo. By applying Loperamide Hydrochloride (LH), the μ-opioid receptor-specific agonist, we established an OIBD-like zebrafish model. Our study found that acetylcholine (ACh) was a key transmitter that derepressed the phenotype induced by LH. Overall, the study showed that the antagonistic role of ACh in the LH-mediated opioid pathway was evolutionarily conserved; moreover, the OIBD-like zebrafish model will be helpful in the future dissection of the molecular pathways involved in gut lumen development and pathology.