Cigarette smoking is a leading cause of preventable mortality worldwide. Nicotine dependence, which reduces the likelihood of quitting smoking, is a heritable trait with firmly established associations with sequence variants in nicotine acetylcholine receptor genes and at other loci. To search for additional loci, we conducted a genome-wide association study (GWAS) meta-analysis of nicotine dependence, totaling 38,602 smokers (28,677 Europeans/European Americans and 9925 African Americans) across 15 studies. In this largest-ever GWAS meta-analysis for nicotine dependence and the largest-ever cross-ancestry GWAS meta-analysis for any smoking phenotype, we reconfirmed the well-known CHRNA5-CHRNA3-CHRNB4 genes and further yielded a novel association in the DNA methyltransferase gene DNMT3B. The intronic DNMT3B rs910083-C allele (frequency=44–77%) was associated with increased risk of nicotine dependence at P=3.7 × 10 −8 (odds ratio (OR)=1.06 and 95% confidence interval (CI)=1.04–1.07 for severe vs mild dependence). The association was independently confirmed in the UK Biobank (N=48,931) using heavy vs never smoking as a proxy phenotype (P=3.6 × 10 −4 , OR=1.05, and 95% CI=1.02–1.08). Rs910083-C is also associated with increased risk of squamous cell lung carcinoma in the International Lung Cancer Consortium (N=60,586, meta-analysis P=0.0095, OR=1.05, and 95% CI=1.01–1.09). Moreover, rs910083-C was implicated as a cis-methylation quantitative trait locus (QTL) variant associated with higher DNMT3B methylation in fetal brain (N=166, P=2.3 × 10 −26 ) and a cis-expression QTL variant associated with higher DNMT3B expression in adult cerebellum from the Genotype-Tissue Expression project (N=103, P=3.0 × 10 −6 ) and the independent Brain eQTL Almanac (N=134, P=0.028). This novel DNMT3B cis-acting QTL variant highlights the importance of genetically influenced regulation in brain on the risks of nicotine dependence, heavy smoking and consequent lung cancer.