Background and PurposeCannabidiol (CBD) represents a promising therapeutic tool for treating cannabis use disorder (CUD). This study aimed to evaluate the effects of CBD on the behavioural and gene expression alterations induced by spontaneous cannabinoid withdrawal.Experimental ApproachSpontaneous cannabinoid withdrawal was evaluated 12 h after cessation of CP‐55,940 treatment (0.5 mg·kg⁻¹ every 12 h, i.p.; 7 days) in C57BL/6J mice. The effects of CBD (5, 10 and 20 mg·kg⁻¹, i.p.) on withdrawal‐related behavioural signs were evaluated by measuring motor activity, somatic signs and anxiety‐like behaviour. Furthermore, gene expression changes in TH in the ventral tegmental area, and in the opioid μ receptor (Oprm1), cannabinoid CB₁ receptor (Cnr1) and CB₂ receptor (Cnr2) in the nucleus accumbens, were also evaluated using the real‐time PCR technique.Key ResultsThe administration of CBD significantly blocked the increase in motor activity and the increased number of rearings, rubbings and jumpings associated with cannabinoid withdrawal, and it normalized the decrease in the number of groomings. However, CBD did not change somatic signs in vehicle‐treated animals. In addition, the anxiogenic‐like effect observed in abstinent mice disappeared with CBD administration, whereas CBD induced an anxiolytic‐like effect in non‐abstinent animals. Moreover, CBD normalized gene expression changes induced by CP‐55,940‐mediated spontaneous withdrawal.Conclusions and ImplicationsThe results suggest that CBD alleviates spontaneous cannabinoid withdrawal and normalizes associated gene expression changes. Future studies are needed to determine the relevance of CBD as a potential therapeutic tool for treating CUD.