While emerging evidence suggests a role for gut bacteria in the pathophysiology of substance use disorder (SUD), studies of their impact on brain and behavioral responses to drugs have been very limited so far. We previously showed that gut dysbiosis enhances the nicotine-induced activation of the mesolimbic system and alters nicotine’s motivational properties in mice. However, the mechanisms linking gut bacteria to the mesolimbic system to influence its response to nicotine remain to be identified. Microbial products are suspected to play a role in this process by influencing gene expression, host immune and glial activation and synaptic signalling. In particular, short-chain fatty acids (SCFAs), the major microbial by-products of dietary fiber fermentation, are considered important targets for understanding the role of the gut microbiome in SUD as they notably regulate the secretion of gut hormones and can cross the blood-brain-barrier and affect epigenetic signaling in the brain. Herein we investigated the role of SCFAs in the consequences of several types of gut dysbiosis on the mesolimbic system function in mice. We notably show that SCFAs supplementation in mice with a gut microbiota depletion rescues the enhanced nicotine-evoked neuronal activation in the ventral tegmental area and the nucleus accumbens shell. We further investigated the contribution of SCFAs-modulation of gut hormones and epigenetics in these effects. Our findings will contribute to identifying the mechanisms by which gut microbiome alterations modulate the brain response to nicotine with the potential to improve our understanding of individual propensity to develop nicotine addiction.