The main goal of this study was to evaluate if the administration of cannabidiol (CBD) regulates behavioral and gene expression alterations induced by spontaneous alcohol withdrawal (SAW) in mice. Increasing doses of ethanol were administered to C57BL/6J male mice for 15 days (2.5, 3 and 3.5 g/kg/12 h, p. o.), and SAW was studied at 6, 12, 24, and 72 h after the last ethanol administration. The efficacy of acute CBD (10, 20, and 40 mg/kg, i. p.) to regulate behavioral changes induced by SAW was explored at 6 h. Gene expression analyses of cannabinoid receptors 1 (Cnr1) and 2 (Cnr2), mu-opioid receptor (Opmr1), and proopiomelanocortin (Pomc) in the nucleus accumbens (NAcc), and Pomc and tyrosine hydroxylase (Th) in the ventral tegmental area (VTA), were carried out by real time-PCR. Pearson correlation was used to identify potential associations between the gene expression data and the anxiety-like behaviors. Biostatistical studies suggest associations between gene expression data and the anxiogenic behaviors in mice exposed to the SAW model and treated with VEH and 40 mg/kg of CBD. Mice exposed to the SAW model showed significant somatic withdrawal signs, anxiety-like behaviors, and remarkable changes in the gene expression of all brain targets at 6 h. CBD dose-dependently normalized the behavioral, somatic withdrawal signs and anxiety-like behaviors and modulated gene expression changes in the NAcc, but not in the VTA. The results of this study suggest that CBD may regulate specific alcohol withdrawal-associated alterations. However, further studies are required to explore the possible mechanisms involved.