Nicotine withdrawal symptoms contribute to relapse in smokers, thereby prolonging the harm caused by smoking. To investigate the molecular basis for this phenomenon, we conducted a genome-wide association study of DSM-IV nicotine withdrawal in a sample of African American (AA) and European American (EA) smokers. A combined AA and EA meta-analysis (n = 8021) identified three highly correlated single nucleotide polymorphisms (SNPs) in the protocadherin (PCDH)-α, -β and -γ gene cluster on chromosome 5 that were associated with nicotine withdrawal (P < 5×10-8). We then studied one of the SNPs, rs31746, in an independent sample of smokers who participated in an intravenous nicotine infusion study that followed overnight smoking abstinence. After nicotine infusion, abstinent smokers with the withdrawal risk allele experienced greater alleviation of their urges to smoke, as assessed by the Brief Questionnaire on Smoking Urges (BQSU). Prior work has shown that the PCDH-α, -β and -γ genes are expressed in neurons in a highly organized manner. We found that rs31746 mapped to a long-range neuron-specific enhancer element shown previously to regulate PCDH-α, -β and -γ gene expression. Using Braincloud mRNA expression data, we identified a robust and specific association between rs31746 and PCDH-β8 mRNA expression in frontal cortex tissue (P < 1 × 10-5). We conclude that PCDH-α, -β and -γ gene cluster regulatory variation influences the severity of nicotine withdrawal. Further studies on the PCDH-α, -β and -γ genes and their role in nicotine withdrawal may inform the development of novel smoking cessation treatments and reduce the harm caused by tobacco smoking.