Background and PurposeAbrupt discontinuation of nicotine, the main psychoactive component in tobacco, induces a withdrawal syndrome in nicotine‐dependent animals, consisting of somatic and affective signs, avoidance of which contributes to drug maintenance. While blockade of fatty acid amide hydrolase, the primary catabolic enzyme of the endocannabinoid arachidonoylethanolamine (anandamide), exacerbates withdrawal responses in nicotine‐dependent mice, the role of monoacylglycerol lipase (MAGL), the main hydrolytic enzyme of a second endocannabinoid 2‐arachidonylglycerol (2‐AG), in nicotine withdrawal remains unexplored.Experimental ApproachTo evaluate the role ofMAGLenzyme inhibition in nicotine withdrawal, we initially performed a genetic correlation approach using theBXDrecombinant inbred mouse panel. We then assessed nicotine withdrawal intensity in the mouse after treatment with the selectiveMAGLinhibitor,JZL184, and after genetic deletion of the enzyme. Lastly, we assessed the association between genotypes and smoking withdrawal phenotypes in two human data sets.Key ResultsBXDmice displayed significant positive correlations between basalMAGLmRNAexpression and nicotine withdrawal responses, consistent with the idea that increased 2‐AGbrain levels may attenuate withdrawal responses. Strikingly, theMAGLinhibitor,JZL184, dose‐dependently reduced somatic and aversive withdrawal signs, which was blocked by rimonabant, indicating aCB1receptor‐dependent mechanism.MAGL‐knockout mice also showed attenuated nicotine withdrawal. Lastly, genetic analyses in humans revealed associations of theMAGLgene with smoking withdrawal in humans.Conclusions and ImplicationsOverall, our findings suggest thatMAGLinhibition maybe a promising target for treatment of nicotine dependence.