Morphine is one of the most severely abused drugs in the world. Previous research on morphine addiction has focused on the central nervous system (CNS). Studies have shown that a two-way regulation of the brain and gut microbiota (GM), suggesting a link between GM and CNS disease. However, the functional mechanism underlying the relationship between intestinal flora and morphine dependence is unclear. In this study, the effect of sinomenine on morphine addiction was evaluated based on the microbiota-gut-brain axis (MGBA). The results show that the GM plays an important role in morphine dependence. Morphine treatment induced zebrafish conditional position preference (CPP), and significantly changed zebrafish GM characteristics and the expression of MGBA-related genes in the zebrafish brain and intestine. Importantly, sinomenine, an alkaloid with a similar structure to morphine, can reverse these morphine-induced changes. Subsequently, morphine-dependent CPP training was performed after antibiotic administration. After antibiotic treatment, zebrafish CPP behavior, the composition and proportions of the zebrafish GM, and the expression of MGBA-related genes in zebrafish were changed. More interestingly, sinomenine was no longer effective in treating morphine dependence, indicating that antibiotic-driven intestinal flora imbalance alters the efficacy of sinomenine on morphine-dependent zebrafish. This study confirms that the MGBA is bidirectionally regulated, highlighting the key role of the GM in the formation and treatment of morphine dependence, and may provide new treatment strategies for using traditional Chinese medicine to treat drug addiction. CI - Copyright (c) 2020 Chen, Zhijie, Yuting, Shilin, Qichun, Jinying, Chaohua, Jing and Zhixian.