Alcohol use disorder (AUD) is known to be influenced by environmental factors. Voluntary physical activity (VPA) has been proven to be rewarding and to play a role in preventing drug relapse. In a previous study, we found that VPA mitigated ethanol-rewarding effects by preventing ethanol-induced conditioned place preference (CPP). There is evidence demonstrating alterations in the neurotrophic signaling of brain-derived neurotrophic factor (BDNF) in mice subjected to both CPP conditioning and VPA. Considering the putative participation of the BDNF signaling in ethanol-CPP and the high-affinity BDNF receptors TrkB 145 kDa and 90 kDa, we sought to address whether the expression of these receptors is changed in the nucleus accumbens (NAc) and prefrontal cortex (PFC) of ethanol-treated mice exposed to VPA. Mice were assigned into four groups. They were housed in home cages with locked (“Sedentary”) or unlocked running wheels (VPA), and treated with saline or 1.8 g/kg ethanol during the conditioning phase. The groups are referred as Saline-Sedentary, Saline-VPA, Ethanol-Sedentary and Ethanol-VPA. TrkB receptors do not play a relevant role in ethanol-induced CPP or in VPA-induced protection against ethanol CPP. VPA and ethanol exposure decreased TrkB 145/90 ratio in the PFC. No differences were found among groups in the NAc. Considering that TrkB 90 kDa can counterbalance the excessive neurotrophic signaling mediated by BDNF-TrkB 145 kDa in the brain, the lower TrkB 145/90 ratio might represent a putative protection to stressful events involving BDNF-induced glutamatergic hyperexcitability and excitotoxicity.