Although numerous studies have revealed significant associations between variants in the nicotinic acetylcholine receptors (nAChR) subunits and nicotine dependence (ND), only few studies were performed in Chinese subjects. Here, we performed association and interaction analysis for 20 single nucleotide polymorphisms (SNPs) in the CHRNB3-CHRNA6 gene cluster with ND in a Chinese Han population (N = 5,055). We found nominally significant associations for all tested SNPs with ND measured by the Fagerström Test for Nicotine Dependence score; of these, 11 SNPs remained significant after Bonferroni correction for multiple tests (p = 9 × 10-4~2 × 10-3). Further conditional analysis indicated that no other SNP was significantly associated with ND independent of the most-highly significant SNP, rs6474414. Also, our haplotype-based association analysis indicated that each haplotype block was significantly associated with ND (p < 0.01). Further, we provide the first evidence of the genetic interaction of these two genes in affecting ND in this sample with an empirical p-value of 0.0015. Finally, our meta-analysis of samples with Asian and European origins for five SNPs in CHRNB3 showed significant associations with ND, with p-values ranging from 6.86 × 10-14 for rs13280604 to 6.50 × 10-8 for rs4950. This represents the first study showing that CHRNB3/A6 are highly associated with ND in a large Chinese Han sample.