Objectives: The aim of the present study was to investigate the effects of treatment with ayahuasca on the rewarding properties of the psychostimulant methylphenidate. Methods: The rewarding properties of ayahuasca (100 mg/kg, orally) and methylphenidate (10 mg/kg, i.p.) were investigated using the conditioned place preference (CPP) model. Furthermore, we evaluated the effects of repeated treatment with ayahuasca on the reinstatement of methylphenidate-induced CPP. Fos expression was evaluated in different limbic structures (cingulate cortex—area 1, prelimbic cortex, infralimbic cortex, orbitofrontal cortex—lateral orbital area, nucleus accumbens core and shell, ventral tegmental area, dorsal striatum, and basolateral amygdala) upon each experimental phase. Results: Both ayahuasca and methylphenidate induced CPP in mice. However, ayahuasca had limited effects on Fos expression, while methylphenidate altered Fos expression in several brain regions associated with the behavioral effects of drugs of abuse. Treatment with ayahuasca after conditioning with methylphenidate blocked the reinstatement of methylphenidateinduced CPP. Those behavioral effects were accompanied by changes in Fos expression patterns, with ayahuasca generally blocking the changes in Fos expression induced by conditioning with methylphenidate and/or reexposure to methylphenidate. Conclusions: Our findings suggest that ayahuasca restored normal brain function in areas associated with the long-term expression of drug wanting/seeking in animals conditioned to methylphenidate.