Heroin is a highly addictive drug that causes axonal damage. Here, manganese-enhanced magnetic resonance imaging (MEMRI) was used to dynamically monitor axonal transport at different stages of heroin addiction. Rat models of heroin addiction (HA) and prolonged heroin addiction (PHA) were established by injecting rats with heroin at different stages. Heroin-induced learning and memory deficits were evaluated in the Morris water maze (MWM), and MEMRI was used to dynamically evaluate axonal transport in the olfactory pathway. The expression of proteins related to axonal structure and function was also assessed by Western blotting. Transmission electron microscopy (TEM) was used to observe ultrastructural changes, and protein levels of neurofilament heavy chain (NF-H) were analyzed by immunofluorescence staining. HA rats, especially PHA rats, exhibited worse spatial learning and memory than control rats. Compared with HA rats and control rats, PHA rats exhibited significantly longer escape latencies, significantly fewer platform-location crossings, and significantly more time in the target quadrant during the MWM test. Mn2+ transport was accelerated in HA rats. PHA rats exhibited severely reduced Mn2+ transport, and the axonal transport rate (ATR) was significantly lower in these rats than in control rats (P < 0.001). The levels of cytoplasmic dynein and kinesin-1 were significantly decreased in the PHA group than in the control group (P < 0.001); additionally, the levels of energy-related proteins, including cytochrome c oxidase (COX) IV and ATP synthase subunit beta (ATPB), were lower in the PHA group (P < 0.001). The brains of heroin-exposed rats displayed an abnormal ultrastructure, with neuronal apoptosis and mitochondrial dysfunction. Heroin exposure decreased the expression of NF-H, as indicated by significantly reduced staining intensities in tissues from HA and PHA rats (P < 0.05). MEMRI detected axonal transport dysfunction caused by long-term repeated exposure to heroin. The main causes of axonal transport impairment may be decreases in the levels of motor proteins and mitochondrial dysfunction. This study shows that MEMRI is a potential tool for visualizing axonal transport in individuals with drug addictions, providing a new way to evaluate addictive encephalopathy.© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.